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1

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

PubMed

Outbreaks of measles and mumps occur regularly in the UK. Rapid diagnosis of acute infection is important for both infection control and epidemiological purposes. The objective of this study was to compare the performance of an automated platform (DiaSorin Liaison(®), Saluggia, Italy) with a manual capture enzyme immunoassay (EIA; Microimmune, Hounslow, UK) for the detection of measles and mumps IgM antibodies in serum from symptomatic individuals. Ninety sera tested previously for measles (n=50) and mumps (n=40) IgM using the manual EIA were tested retrospectively using the DiaSorin Liaison(®) and the results compared. Sensitivity, specificity, inter-assay variability and intra-assay variability of the Liaison(®) assays were calculated. Sensitivity and specificity of the Liaison(®) assay for measles IgM were 92% and 100% respectively, with inter-assay variation of 14.1% and intra-assay variation of 12.5%. The sensitivity and specificity of the mumps IgM Liaison(®) assay were 88% and 95% respectively, with an inter-assay and intra-assay variation of 13.9% and 5.3% respectively. Both the measles and mumps IgM Liaison(®) assays gave fewer equivocal results than the EIA. Neither Liaison(®) IgM assay showed any cross-reactivity with sera positive against other viruses, however the measles IgM EIA cross-reacted with parvovirus IgM. The automated Liaison(®) assays are more specific, cheaper and less labour-intensive compared to the manual EIA. The Liaison(®) assays benefit from reduced number of equivocal results compared to the EIA for both measles and mumps IgM. This allows clinical decisions to be made accurately and in a timely manner. PMID:24183918

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

2014-02-01

2

Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae.  

PubMed Central

An enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this EIA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection. PMID:1583120

Uldum, S A; Jensen, J S; S?ndergard-Andersen, J; Lind, K

1992-01-01

3

Evaluations of Commercial West Nile Virus Immunoglobulin G (IgG) and IgM Enzyme Immunoassays Show the Value of Continuous Validation  

Microsoft Academic Search

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and

Annette K. Malan; Thomas B. Martins; Harry R. Hill; Christine M. Litwin

2004-01-01

4

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

PubMed

The aim of the study was to compare reactivity in the Mercia immunoglobulin M enzyme immunoassay (IgM EIA) and the Venereal Disease Research Laboratory (VDRL) after treatment of 229 previously untreated patients with early syphilis. At three months, the VDRL and the IgM EIA were negative in 41 (38%) and 71 (62%) cases, respectively; a four-fold or greater decrease in VDRL titre occurred in 106 (99%). At six months, the VDRL and the IgM EIA were negative in 45 (48%) and 69 (71%) patients, respectively; a four-fold or greater decrease in VDRL titre occurred in 88 (95%) and an eight-fold or greater decrease in 80 (86%). At 12 months, the VDRL and the IgM EIA were negative in 35 (70%) and 55 (92%) patients, respectively; a four-fold or greater decrease in VDRL titre occurred in 49 (98%) and an eight-fold or greater decrease in 47 (94%). The Mercia IgM EIA is as sensitive as the VDRL in monitoring treatment of primary syphilis but not as sensitive as the finding of a four-fold or eight-fold decrease in VDRL titre in patients treated for secondary or early latent infection. PMID:18824622

McMillan, A; Young, H

2008-10-01

5

Enzyme-linked immunoassay for dengue virus IgM and IgG antibodies in serum and filter paper blood  

Microsoft Academic Search

BACKGROUND: The reproducibilty of dengue IgM and IgG ELISA was studied in serum and filter paper blood spots from Vietnamese febrile patients. METHODS: 781 pairs of acute (t0) and convalescent sera, obtained after three weeks (t3) and 161 corresponding pairs of filter paper blood spots were tested with ELISA for dengue IgG and IgM. 74 serum pairs were tested again

Thanh Nga T Tran; Peter J de Vries; Lan Phuong Hoang; Giao T Phan; Hung Q Le; Binh Q Tran; Chi Mai T Vo; Nam V Nguyen; Piet A Kager; Nico Nagelkerke; Jan Groen

2006-01-01

6

An enzyme immunoassay for plasma betamethasone  

SciTech Connect

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

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

1981-03-01

7

Gold bands as a suitable surface for enzyme immunoassays.  

PubMed

Gold bands sputtered over a polymeric material, Kapton, are employed for the development of enzyme immunoassays. The immunological interaction takes place between human IgM and alkaline phosphatase (AP) conjugated anti-IgM. The model analyte (IgM) could be determined following a non-competitive design in the range of 0.05-5 ppm, with a limit of detection of 50 ppb. After the interaction, gold bands are sequentially inserted in a flow system and the extension of the reaction is followed through the enzymatic hydrolysis of naphthylphosphate, AP substrate. The product, naphthol, is oxidised to naphtoquinone in the gold band of the flow cell that constitutes the detector. Parameters affecting the interaction are studied and calibration curves are performed. The reproducibility between different bands (RSD=4%, n=5) and possibilities of regeneration are also detailed. PMID:12191928

Abad-Villar, Eva M; Fernández-Abedul, M Teresa; Costa-García, Agustín

2002-09-01

8

Rapid Diagnosis of Japanese Encephalitis by Using an Immunoglobulin M Dot Enzyme Immunoassay  

Microsoft Academic Search

Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field

TOM SOLOMON; THI THU THAO; NGUYEN MINH DUNG; RACHEL KNEEN; ANANDA NISALAK; DAVID W. VAUGHN; JEREMY FARRAR; TRAN TINH HIEN; NICHOLAS J. WHITE; MARY JANE CARDOSA

1998-01-01

9

Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates  

DOEpatents

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.

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

1997-11-25

10

Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates  

DOEpatents

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.

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

1997-01-01

11

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

PubMed Central

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

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

2013-01-01

12

Validation of an enzyme immunoassay for serodiagnosis of acute Q fever  

Microsoft Academic Search

An enzyme immunoassay was validated for the serodiagnosis of acute Q fever. Minimum positive tests were determined for both serial dilutions and a single dilution of patient sera. To establish the specificity of the test, 152 serum samples were tested from individuals with no evidence of pastCoxiella burnetii infection. Diagnostic titers were set at ?128 for the IgM and IgG

D. Waag; J. Chulay; T. Marrie; M. England; J. Williams

1995-01-01

13

Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein  

Microsoft Academic Search

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and\\/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where

DARIO SOLDATESCHI; GRAZIA MARIA DAL MASO; MARCELLO VALASSINA; LAURA SANTINI; SILVIA BIANCHI; MARIA GRAZIA CUSI

1999-01-01

14

Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).  

PubMed

This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors. PMID:16179424

Lequin, Rudolf M

2005-12-01

15

Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics.  

PubMed

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ? 95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

Porwancher, Richard B; Hagerty, C Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J B; Kopnitsky, Mark; Steere, Allen C; Kulas, Karen; Wong, Susan J

2011-05-01

16

Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics ?  

PubMed Central

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ?95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

Porwancher, Richard B.; Hagerty, C. Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J. B.; Kopnitsky, Mark; Steere, Allen C.; Kulas, Karen; Wong, Susan J.

2011-01-01

17

Application of enzyme immunoassays for testing haemocompatibility of biomedical polymers  

Microsoft Academic Search

In this study enzyme immunoassays are presented for the assessment of platelet adhesion\\/activation and fibrinogen adsorption\\/conformation. The estimation of platelet adhesion and activation was performed with two enzyme immunoassays (EIAs) using monoclonal antibodies directed against CD42b (GP Ib) and CD 62 (GMP 140 or P-Selectin). The applicability of EIA was first demonstrated in microtitre plates coated with fibrinogen. The thrombogenic

Th. Groth; E. J. Campbell; K. Herrmann; B. Seifert

1995-01-01

18

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...

19

Possible false-negative results on therapeutic drug monitoring of phenytoin using a particle enhanced turbidimetric inhibition immunoassay in a patient with a high level of IgM.  

PubMed

: In this report, the authors described the unusual case of a patient in whom the plasma phenytoin concentration was unexpectedly not detected on a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) technique, a typical immunoassay for phenytoin. The plasma concentration was measured using PETINIA and high-performance liquid chromatography in a 69-year-old male patient treated with fosphenytoin intravenously at the standard dose for 7 days. Although the plasma concentration of phenytoin was below the limit of detection (<0.5 mcg/mL) on PETINIA after the administration of fosphenytoin, the trough plasma concentration was estimated to be between 5 and 10 mg/L on high-performance liquid chromatography. When the plasma concentrations of IgM and IgG were measured using an enzyme-linked immunosorbent assay, the plasma IgG level was within the reference range, whereas the plasma IgM level was 2-3 times higher than the upper limit of the reference range. We concluded that the PETINIA method yielded a possible false-negative result regarding the phenytoin level in this patient, perhaps because of some hindrance to the measurement process by IgM. This case suggests that false-negative results should be considered when therapeutic drug monitoring reveals abnormally low values using PETINIA and that it is necessary to evaluate the plasma IgM level. PMID:24632808

Hirata, Kenshiro; Saruwatari, Junji; Enoki, Yuhuki; Iwata, Kazufumi; Urata, Yukino; Aizawa, Keiji; Ueda, Kentaro; Shirouzono, Takumi; Imamura, Motoki; Moriuchi, Hiroshi; Ishima, Yu; Kadowaki, Daisuke; Watanabe, Hiroshi; Hirata, Sumio; Maruyama, Toru; Fukunaga, Eiko

2014-10-01

20

Development of enzyme immunoassay for detection of DDT  

Microsoft Academic Search

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p?-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions,

Masashi Hirano; Kazuyuki Kitamura; Ikuo Kato; Chizuko Yanaihara; Ken-Ichi Iwamoto; Makiko Sekiyama; Chiho Watanabe; Takashi Nakamoto; Nobukazu Miyamoto; Yuta Onishi; Koji Arizono

2008-01-01

21

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...

22

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 ...

23

Enzyme-Labeling of Antibodies and Their Fragments for Enzyme Immunoassay and Immunohistochemical Staining  

Microsoft Academic Search

The use of an enzyme as a label has a number of advantages over the use of other labels in both immunohistochemistry and immunoassay. Immunofluorescence techniques are not suitable for ultrastructural research on cells, and ferritin-labeled antibodies allow only electronmicroscopic studies. By contrast, enzyme-labeled antibodies permit localization of cellular antigens in relation to tissue structures under light microscope and also

Eiji Ishikawa; Masayoshi Imagawa; Seiichi Hashida; Shinji Yoshitake; Yoshitaka Hamaguchi; Tetsuo Ueno

1983-01-01

24

The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten  

Microsoft Academic Search

BackgroundIn hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays.

Anupam Basu; Seema Nara; Shail K. Chaube; Kiran Rangari; Kiran P. Kariya; Tulsidas G. Shrivastav

2006-01-01

25

Matrix effects on an antigen immobilized format for competitive enzyme immunoassay of salivary testosterone  

Microsoft Academic Search

Matrix interferences in salivary testosterone enzyme immunoassays are important in the development of direct ELISA. An alternative format for sensitive enzyme immunoassay of testosterone was developed using immobilization of the antigen as part of a protein conjugate using oligoethylene glycol linker to project the antigen, with color development via enzyme labeled secondary antibody. This technique gave the required sensitivity for

John S. Mitchell; Tim E. Lowe

2009-01-01

26

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

PubMed

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

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

1984-03-01

27

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay  

Microsoft Academic Search

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime–bovine serum albumin (F-3-O-CMO-BSA) was used as

Seema Nara; Vinay Tripathi; Shail K. Chaube; Kiran Rangari; Harpal Singh; Kiran P. Kariya; Tulsidas G. Shrivastav

2008-01-01

28

Evaluation of Four Commercial IgG- and IgM-specific Enzyme Immunoassays for Detecting Mycoplasma pneumoniae Antibody: Comparison with Particle Agglutination Assay  

PubMed Central

Diagnosis of Mycoplasma pneumoniae infection is important due to its variable clinical manifestations and absence of response to beta-lactams. Introduction of enzyme immunoassays (EIAs) for serologic diagnosis of M. pneumoniae has made it possible to separate the analyses of specific IgG and IgM antibodies. We compared four different commercial EIAs, ImmunoWELL IgG, IgM (GenBio), Medac IgG, IgA, IgM (Medac), Platelia IgG, IgM (Sanofi Pasteur), and Ridascreen IgG, IgA, IgM (r-Biopharm) with indirect particle agglutination assay (PA), Serodia-MycoII (Fujirebio). We tested 91 specimens from 73 pediatric patients (2-17 yr) hospitalized at a tertiary-care hospital between December 2005 and January 2006. The measurements of IgM EIAs were correlated with PA titers (Spearman's correlation coefficient, from 0.89 to 0.92) with high concordance rates, ranging from 82.4% to 92.3%. However, some negative IgM-EIA results in PA-positive specimens indicated that serial samplings with convalescent sera would be necessary to confirm M. pneumoniae infection. PMID:17982225

Yoo, Soo Jin; Oh, Hye-Jeon

2007-01-01

29

Homogeneous enzyme immunoassay for pyrethroid pesticides and their derivatives using bacillary alpha-amylase as label  

Microsoft Academic Search

A new enzyme immunoassay for detection of some pyrethroid pesticides (permethrin, phenothrin) and their derivatives containing 3-phenoxybenzoic group has been developed. It is based on enzyme multiplied immunoassay technique (EMIT), modulation of catalytic activity of hapten-enzyme conjugate by anti-hapten antibodies and restoring the initial activity level by free hapten in the sample tested. Alpha-amylase from Bacillus subtilis is proposed as

Anatoliy V. Zherdev; Boris B. Dzantiev; Janna N. Trubaceva

1997-01-01

30

Immunoassays  

NASA Astrophysics Data System (ADS)

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.

Hsieh, Y.-H. Peggy

31

Enzyme immunoassay of thyroxin with a centrifugal analyzer  

SciTech Connect

We have applied a homogeneous enzyme immunoassay for determination of thyroxinin serum to the ''Cobas Bio'' centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing ''Lipex,'' an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37/sup 0/C. To 20 ..mu..L of sample mixture is added 125 ..mu..L of reagent (thyroxin antibodies and NAD/sup +/) and this mixture is incubated for 10 s. Then 25 ..mu..L of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 ..mu..g of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.

Izquierdo, J.M.; Sotorrio, P.; Quiros, A.

1982-01-01

32

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

PubMed

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

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

2001-07-01

33

Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.  

PubMed

Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (p<0.0001) lower reactivity was demonstrated in samples from previously infected patients where acute rubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms. PMID:24487099

van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia

2014-04-01

34

Detection of Eastern Equine Encephalomyelitis Viral Antigen in Avian Blood by Enzyme Immunoassay: A Laboratory Study.  

National Technical Information Service (NTIS)

An enzyme immunoassay (EIA) was evaluated for its efficacy at detecting eastern equine encephalomyelitis (EEE) virus in avian blood and brain specimens. Preliminary analysis of blood from experimentally infected house sparrows and naturally infected whoop...

T. W. Scott, J. G. Olson

1986-01-01

35

HIV-Selectest enzyme immunoassay and rapid test: ability to detect seroconversion following HIV-1 infection.  

E-print Network

??HIV-Selectest is a serodiagnostic enzyme immunoassay (EIA), containing p6 and gp41 peptides, designed to differentiate between vaccine-induced antibodies and true infections. A rapid test version… (more)

Golding, Hana.

2010-01-01

36

Rapid dioxin screening of milk and water by enzyme immunoassay  

SciTech Connect

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.

Harrison, R.O. [ImmunoSystems Inc., Scarborough, ME (United States); Carlson, R.E. [Ecochem Research, Inc., Chaska, MN (United States); Shirkhan, H. [Fluid Management Systems, Inc., Atlanta, GA (United States)

1995-12-01

37

Enzyme immunoassay for mycophenolic acid in milk and cheese.  

PubMed

Mycophenolic acid (MPA) was reacted with N-hydroxysuccinimide and conjugated to keyhole limpet hemocyanin (KLH), and to horseradish peroxidase (HRP), respectively. The MPA-KLH was used to produce anti-MPA antiserum in rabbits. A competitive direct enzyme immunoassay (EIA) for MPA was established with anti-MPA antiserum and MPA-HRP conjugate. The mean 50% inhibition and detection limit of MPA standard curves (n = 103) were 197 +/- 67 and 81 +/- 48 pg/mL, respectively. The EIA was specific for MPA and its synthetic 2-morpholinoethyl ester, mycophenolate mofetil (91% relative cross-reactivity). Raw bulk milk and pasteurized milk, with and without beta-glucuronidase pretreatment, were analyzed by EIA. No MPA was found in milk, at a detection limit of 100 pg/mL (recovery 58-66% at 0.125-2 ng/mL). Blue-veined cheese from the German market (n = 53) was analyzed by EIA, and the detection limit was at 0.5 ng/g (recovery 68-79% at 5-100 ng/g). All but two cheeses contained MPA, although mostly (66%) at levels of <10 ng/g. MPA at 400-1200 ng/g was found in Roquefort cheeses. Highest levels (4-11 microg/g) were found in a German soft cheese preparation. MPA levels in mycelium-rich parts of cheese were 3 times higher than in mycelium-free parts. PMID:18611027

Usleber, Ewald; Dade, Melanie; Schneider, Elisabeth; Dietrich, Richard; Bauer, Johann; Märtlbauer, Erwin

2008-08-27

38

Bioluminescent Enzyme Immunoassay for the Detection of Norovirus Capsid Antigen  

PubMed Central

An ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y = 0.66x ? 3.21, r = 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 105 to 106 copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis. PMID:23081816

Ohiro, Yoshiyuki; Ito, Mitsuki; Makinodan, Mitsuru; Ohta, Tsubasa; Suzuki, Wataru; Takayasu, Susumu; Tsuge, Harufumi

2012-01-01

39

Incorporation of Different Bridge Length Linkers in Enzyme and Its Use in the Preparation of Enzyme Conjugates for Immunoassay  

Microsoft Academic Search

An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6?amino caproic acid (6ACA) followed by ADH. The different bridge length linkers?incorporated

Tulsidas G. Shrivastav

2005-01-01

40

Assessment of commercial enzyme immunoassay for hepatitis C virus serotyping.  

PubMed Central

AIMS: To assess a commercial enzyme immunoassay (EIA) for the serotyping of hepatitis C virus (HCV) for routine use in a diagnostic laboratory setting, as well as for noting the serotype prevalence of selected specimens. METHODS: Seventy six serum specimens, submitted to the laboratory for routine hepatitis studies between May 1992 and February 1996 and stored at -20 degrees C, were evaluated. These specimens were categorised into specific hepatic, renal, and paediatric clinical conditions. The specimens all tested positive for HCV antibodies on a screening EIA, with confirmation on a recombinant immunoblot assay (RIBA). Certain specimens were also HCV RNA positive by the reverse transcription polymerase chain reaction (RT-PCR). All the specimens were serotyped using the newly developed serotyping EIA. RESULTS: Twenty seven (35.5%) specimens were typable. Type 5 predominated (56%), followed by type 1 (33%), types 1 and 6 (7%) and type 3 (4%). The serotype 5 specimens showed 85% and 90% reactivity with recombinant antigens c100-3 and c22-3c, respectively; serotype 1 specimens showed 75% and 100% reactivity with these antigens. All serotype 5 specimens reacted with the c33-c antigen, but only 60% of serotype 1 specimens reacted with this antigen. The differences in the reactivity of the serotype 5 and serotype 1 specimens for c33-c antigen in the RIBA were significant, but no significant differences in reactivity for antigens c-1-1, c100-3, and c22-3 were noted. Serotype 3 specimens showed equal reactivity with all four antigens used in the RIBA. CONCLUSION: The serotyping EIA was easy to use, rapid, and cost effective compared with molecular assays. This assay seems to be ideal for the routine diagnostic laboratory setting, but could not be used for certain clinical specimens. The demonstration of serotypes 5, 1, and 3 was not unexpected in this cohort. The occurrence of serotype 6, although concurrent and more likely to be a false cross reaction with serotype 1 peptides, requires confirmation by molecular genotyping before it can be claimed that this type is present in South Africa. PMID:9038737

Webber, L M; Els, S; Taylor, M B; Grabow, W O

1996-01-01

41

Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides  

SciTech Connect

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)

Krogstad, D.J. (Barnes Hospital, St. Louis, MO); Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

1981-07-01

42

Assignment of the gene for uroporphyrinogen decarboxylase to human chromosome 1 by somatic cell hybridization and specific enzyme immunoassay  

Microsoft Academic Search

A specific enzyme immunoassay of uroporphyrinogen decarboxylase was developed and applied to the detection of the human enzyme in man-rodent somatic cell hybrids. This method allowed to assign the gene for uroporphyrinogen decarboxylase to human chromosome 1.

H. de Verneuil; B. Grandchamp; Chantal Foubert; Dominique Weil; Cong N'Guyen; Marie-Sylvie Gross; Shigeru Sassa; Y. Nordmann

1984-01-01

43

Buprenorphine detection in urine using liquid chromatography-high-resolution mass spectrometry: comparison with cloned enzyme donor immunoassay (ThermoFisher) and homogeneous enzyme immunoassay (immunalysis).  

PubMed

A sensitive liquid chromatographic-high-resolution mass spectrometric (LC-HR-MS) assay for buprenorphine and its urinary metabolites has been developed that requires minimal sample preparation. The results obtained have been compared with those given by (i) cloned enzyme donor immunoassay (CEDIA) and (ii) homogeneous enzyme immunoassay (HEIA) in the analysis of patient urines submitted for buprenorphine analysis. Centrifuged urine (100 µL) was diluted with internal standard solution (25 µL) + LC eluent (875 µL), and 50 µL of the prepared sample were analyzed (Accucore Phenyl-Hexyl column). MS detection was in alternating positive and negative mode using heated electrospray ionization (ThermoFisher Q Exactive). Intra- and inter-assay accuracy and precision were 104-128 and <11%, respectively, at 5 µg/L. Limits of detection were 1.3 µg/L (buprenorphine, norbuprenorphine and buprenorphine glucuronide) and 2.5 µg/L (norbuprenorphine glucuronide). Immunoassay sensitivity and selectivity were 97 and 100% (HEIA) and 99 and 84% (CEDIA), respectively, compared with LC-HR-MS. In 120 patient urines, norbuprenorphine glucuronide was easily the most abundant analyte except when adulteration with buprenorphine had occurred. The median immunoreactive buprenorphine species present (unhydrolysed urine) were 7.5 and 13% for HEIA and CEDIA, respectively. However, codeine, dihydrocodeine, morphine and morphine-3-glucuronide did not interfere in the HEIA assay. PMID:24925983

Belsey, Sarah L; Couchman, Lewis; Flanagan, Robert J

2014-09-01

44

HIV-Selectest Enzyme Immunoassay and Rapid Test: Ability To Detect Seroconversion following HIV-1 Infection?  

PubMed Central

HIV-Selectest is a serodiagnostic enzyme immunoassay (EIA), containing p6 and gp41 peptides, designed to differentiate between vaccine-induced antibodies and true infections. A rapid test version of the HIV-Selectest was developed. Both assays detected HIV antibodies in men and women within 2 to 4 weeks of infection, with sensitivity similar to third-generation EIAs. PMID:19906903

Khurana, Surender; Norris, Philip J.; Busch, Michael P.; Haynes, Barton F.; Park, Susan; Sasono, Pretty; Mlisana, Koleka; Salim, Abdool Karim; Hecht, Frederick M.; Mulenga, Joseph; Chomba, Elwyn; Hunter, Eric; Allen, Susan; Nemo, George; Rodriguez-Chavez, Isaac R.; Margolick, Joseph B.; Golding, Hana

2010-01-01

45

ENZYME-LINKED IMMUNOASSAYS FOR THE DETECTION OF MICROBIAL ANTIGENS AND THEIR ANTIBODIES  

EPA Science Inventory

The advantages of enzyme-immunoassay (EIA) over radioactive assay techniques are mainly convenience in use, in that the labelled immunoreagents are stable for long periods, and the precautions and disposal procedures required for radioisotopes are unnecessary. In addition, the us...

46

Sensitive detection of a plant virus by electrochemical enzyme-linked immunoassay  

Microsoft Academic Search

The electrochemical enzyme-linked immunoassay increases the sensitivity of the detection of cucumber mosaic virus (CMV) by\\u000a 5-fold compared with the spectrophotometric o-phenylenediamine (OPD) enzyme-linked immunosorbent assay (ELISA). The detection\\u000a limit for the purified CMV is 1.0 ng\\/mL and the highest dilution ratio of the infected leaf sap is 1?:?5.0 × 104. The method is based on coupling the oxidation reaction

K. Jiao; Wei Sun; Shu-Sheng Zhang

2000-01-01

47

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay.  

PubMed

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody. PMID:18023401

Nara, Seema; Tripathi, Vinay; Chaube, Shail K; Rangari, Kiran; Singh, Harpal; Kariya, Kiran P; Shrivastav, Tulsidas G

2008-02-01

48

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

PubMed Central

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

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

1988-01-01

49

Evaluation of an Enzyme Immunoassay Technique for Detection of Antibodies against Treponema pallidum  

Microsoft Academic Search

In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of Treponema pallidum immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a

Rita Castro; Emília S. Prieto; Irene Santo; Jacinta Azevedo; L. Exposto

2003-01-01

50

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.  

PubMed Central

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added. PMID:337898

Saunders, G C; Bartlett, M L

1977-01-01

51

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

Microsoft Academic Search

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

M. A. Farrington; W. C. Hymer

1987-01-01

52

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

SciTech Connect

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)

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

1980-01-01

53

Detection of Giardia lamblia and Entamoeba histolytica in Stool Samples by Two Enzyme Immunoassays  

Microsoft Academic Search

Two commercially produced enzyme immunoassays (EIAs) to detect antigens of Giardia lamblia and Entamoeba histolytica in stool specimens were evaluated. A total of 276 stool specimens were collected from patients who presented with various\\u000a medical complaints in the outpatient clinic of the Department of Infectious Diseases and Tropical Medicine, University of\\u000a Munich. Every specimen was examined by conventional microscopy and

M. Schunk; T. Jelinek; K. Wetzel; H. D. Nothdurft

2001-01-01

54

USING A COMMERCIALLY AVAILABLE ENZYME IMMUNOASSAY TO QUANTIFY TESTOSTERONE IN AVIAN PLASMA  

Microsoft Academic Search

Using a commercially available testos- terone enzyme immunoassay (EIA), we developed and validated an assay procedure for determining testosterone levels in small-volume (20 mL) avian plasma samples. We evaluated this EIA's utility by measuring plasma testosterone levels in Mourning Doves (Zenaida macroura), White-eyed Vireos (Vireo griseus), Red-eyed Vireos (Vireo olivaceus), and Indigo Buntings (Passerina cyanea). Standard bio- chemical validations (e.g.,

BRIAN E. WASHBURN; JOSHUA J. MILLSPAUGH; DANA L. MORRIS; JOHN H. SCHULZ; JOHN FAABORG

2007-01-01

55

Multilayers enzyme-coated carbon nanotubes as biolabel for ultrasensitive chemiluminescence immunoassay of cancer biomarker  

Microsoft Academic Search

A novel and ultrasensitive chemiluminescence immunoassay (CLIA) method based on multiple enzyme layers assembled multiwall carbon nanotubes (MWCNTs) as signal amplification labels was developed by employing luminol–H2O2-HRP-bromophenol blue (BPB) enhanced chemiluminescence (CL) system for the detection of a cancer biomarker in human serum samples, as exemplified by the measurement of alpha-fetoprotein (AFP) as a model protein. In this study, horseradish

Sai Bi; Hong Zhou; Shusheng Zhang

2009-01-01

56

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

PubMed Central

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

Kurstak, Edouard

1985-01-01

57

Evaluation of three commercial enzyme immunoassay kits for detecting faecal Clostridium difficile toxins  

Microsoft Academic Search

The detection of faecal cytotoxicity using tissue culture was compared with three commercial Clostridium difficile enzyme immunoassay (EIA) kits; Premier C difficile toxin A (Meridian Diagnostic, Inc.); CD-TOX C difficile toxin A (Porton Cambridge); and Cytoclone A+B EIA (Cambridge Biotech Corporation). Of 160 faecal samples examined by all four methods, 52 (32.5%) were cytotoxic, 44 (27.5%) were positive by Premier,

S A Arrow; L Croese; R A Bowman; T V Riley

1994-01-01

58

Development of a Simple and Highly Sensitive Enzyme Immunoassay for Hepatitis C Virus Core Antigen  

Microsoft Academic Search

A highly sensitive enzyme immunoassay (EIA) for the hepatitis C virus (HCV) core antigen (HCVcAg) was developed, and its performance was compared with that of the AMPLICOR HCV test (Roche Molecular Systems). The developed one-step pretreatment method, 30-min incubation of the specimen with a solution containing three different types of detergents (Triton X-100, 3-((3-cholamidopropyl)-dimethylammonio)-1- propanesulfonate (CHAPS), and sodium dodecyl sulfate),

KATSUMI AOYAGI; CHIHARU OHUE; KUMIKO IIDA; TATSUJI KIMURA; EIJI TANAKA; KENDO KIYOSAWA; SHINTARO YAGI

1999-01-01

59

Enzyme Immunoassay for Clenbuterol, an ?2Adrenergic Stimulant  

Microsoft Academic Search

A sensitive double antibody and heterologous enzyme immune-assay for the quantitation of clenbuterol is established. Specific antiserum to this agent was raised in rabbits by immunization with diazotized clenbuterol and human serum albumin conjugate. For competitive reactions, antibody was incubated with a mixture of diazotized clenbuterol analog (NA 1141) labelled with ?-D-galactosidase and unlabelled standard or sample clenbuterol. The antibody-bound

Itaru Yamamoto; Kohji Iwata

1982-01-01

60

Detection of Escherichia coli in wastewater based on enzyme immunoassay  

Microsoft Academic Search

This research describes a fast detection method on the basis of enzyme-linked immunosorbent assay (ELISA) for Escherichia coli in drainage of wastewater treatment plants. Optimized conditions such as the reaction format (sandwich or direct), the concentrations\\u000a of diluted horseradish peroxidase (HRP)-E. coli conjugate, and anti-HPR antibody and pretreatment of E. coli were studied. Those results showed that the linear range

Haiyan Xi; Qiang Cai; Miao He; Hanchang Shi

2007-01-01

61

Comparative analysis of different enzyme immunoassays for assessment of phosphatidylserine-dependent antiprothrombin antibodies.  

PubMed

Phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) were strongly correlated with the presence of lupus anticoagulant showing a high specificity for the diagnosis of antiphospholipid syndrome. However, the main criticism for the clinical applicability of aPS/PT testing is the lack of reproducibility of the results among laboratories. In this study, we measured IgG and IgM aPS/PT using our original in-house enzyme-linked immunosorbent assays (ELISA) and commercial ELISA kits to assess the assay performance and to evaluate the accuracy of aPS/PT results. The study included 111 plasma samples collected from patients and stored at our laboratory for aPS/PT assessment. Sixty-one samples were tested for IgG aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite™ aPS/PT IgG ELISA kit (INOVA Diagnostics, Inc., USA). Fifty samples were evaluated for IgM aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite™ aPS/PT IgM ELISA kit (INOVA Diagnostics). Ninety-eight percent of samples yielded concordant results for IgG aPS/PT and 82 % for IgM aPS/PT. There was an excellent agreement between the IgG aPS/PT assays (Cohen ? = 0.962) and moderate agreement between the IgM aPS/PT assays (? = 0.597). Statistically significant correlations in the aPS/PT results were obtained from both IgG and IgM aPS/PT assays (r = 0.749, r = 0.622, p < 0.001, respectively). In conclusion, IgG and IgM detection by ELISA is accurate. The performance of aPS/PT is reliable, and concordant results can be obtained using different ELISA methods. PMID:24497039

Amengual, Olga; Horita, Tetsuya; Binder, Walter; Norman, Gary L; Shums, Zakera; Kato, Masaru; Otomo, Kotaro; Fujieda, Yuichiro; Oku, Kenji; Bohgaki, Toshiyuki; Yasuda, Shinsuke; Atsumi, Tatsuya

2014-09-01

62

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

Microsoft Academic Search

Two artificial antigens, N\\u000a ?N?-di(O, O-diisopropyl) phosphoryl l-lysine (DIP)-bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Antibodies\\u000a against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition\\u000a enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be\\u000a inhibited by sarin as low

Yong-xin Zhou; Qing-jin Yan; Yun-xiang Ci; Zhen-quan Guo; Kang-Tai Rong; Wen-bao Chang; Yu-fen Zhao

1995-01-01

63

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

NASA Technical Reports Server (NTRS)

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.

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

1987-01-01

64

Enzyme Immunoassay versus Latex Agglutination Cryptococcal Antigen Assays in Adults with Non-HIV-Related Cryptococcosis.  

PubMed

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

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

2014-12-01

65

Enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide triazophos.  

PubMed

A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, which was based on the anti-THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu-HRP). Several components of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p-iodo-phenol, 0.625 mM of luminol, and 4 mM of H(2)O(2) had the best performance. Based on the study of CES, the influence of several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity for detection, IC(50) value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mL and the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot, apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison between the methods of CLEIA and high-performance liquid chromatography-tandem mass spectrum (HPLC-MS/MS) was performed. The results obtained from CLEIA were in agreement with those of HPLC-MS/MS. PMID:22490114

Jin, Maojun; Shao, Hua; Jin, Fen; Gui, Wenjun; Shi, Xiaomei; Wang, Jing; Zhu, Guonian

2012-05-01

66

Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates.  

PubMed

A solid-phase enzyme-linked immunoassay using a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was developed. Antibodies were covalently linked to glutaraldehyde-activated 96-well aminopolystyrene plates. Antigens from test samples were adsorbed to the solid phase and detected using antibodies conjugated with E. coli beta-galactosidase. Glutaraldehyde, N-succinimidyl-3-(2-pyridyldithio)-propionate or N-succinimidyl-6-(4-azido-2-nitrophenylamino)-hexanoate were used as linkers between antibodies and the enzyme. The measurement of fluorescence can be automated for rapid screening of many specimens. The sensitivity limit of the test for HBsAg is about 5-10 pg. PMID:6795220

Neurath, A R; Strick, N

1981-10-01

67

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

PubMed

Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market. PMID:12778970

Kumari, G Lakshmi; Dhir, Ravindra N

2003-01-01

68

Comparison of three enzyme immunoassays for measuring 17beta-estradiol in flushed dairy manure wastewater.  

PubMed

Natural steroidal estrogens are an environmental concern because low nanogram per liter concentrations in water can adversely affect aquatic vertebrate species by disrupting the normal function of their endocrine systems. There is a critical need to accurately measure estrogens in dairy wastes, a potential source of estrogens such as 17beta-estradiol, to assess the risk of estrogen contamination of agricultural drainage waters resulting from land application. Commercially available enzyme immunoassay (EIA) kits have been used for measuring 17beta-estradiol in livestock manure, but it is not known if different EIAs provide similar results. We compared three EIAs by measuring 17beta-estradiol in two samples of flushed dairy manure wastewater (FDMW). The measured concentrations of 17beta-estradiol in FDMW differed according to the immunoassay used. The differences were attributed to a matrix interference associated with coextracted humic substances. Future research should develop methods that enable routine measurement of 17beta-estradiol in livestock wastes by more conclusive analytical techniques such as gas chromatography-mass spectrometry. PMID:15356254

Hanselman, Travis A; Graetz, Donald A; Wilkie, Ann C

2004-01-01

69

Development of an enzyme immunoassay for the detection of trinitrotoluene (TNT) in soil and water  

SciTech Connect

An enzyme immunoassay (EIA) has been developed which is capable of detecting as little as 0.5 ppb TNT in water and 0.25 ppm TNT in soil. Rabbit polyclonal antibodies were raised against a TNT-derivative coupled to bovine serum albumin. These antibodies are coated on both polystyrene tubes and 96-well microtiter plates. Water samples or soil extracts together with a TNT-horseradish peroxidase conjugate are then added and compete for antibody binding sites. Excess conjugate is washed away in a simple rinse step, and substrate (3,3{prime},5,5{prime}-tetramethylbenzidine) is added; the amount of color developed is inversely proportional to the concentration of TNT in the sample. This competitive ELA is relatively specific: 4-amino-2,6-DNT and 2,6-DNT are only 20% as reactive as TNT, and 2,4-DNT shows approximately 1% cross-reactivity. Other common explosives and important metabolites, such as RDX, HMX, nitroglycerin, and dichlorobenzoic acids, are essentially non-reactive in this assay. The microliter plate-based assay is complete in 90 minutes and is suitable for laboratory use. The tube-based test is run in less than 30 minutes and is field-portable. The soil extraction protocol employed for this assay utilizes methanol as the solvent and can be completed in 10 minutes. A simple dilution step is the only sample preparation that is required before running soil extracts in the immunoassay.

Larkin, K.A.; Ferguson, B.S.; Matt, J.J.; Fan, T.S. [ImmunoSystems Inc., Scarborough, ME (United States)

1995-12-31

70

Fluorescent artificial enzyme-linked immunoassay system based on Pd/C nanocatalyst and fluorescent chemodosimeter.  

PubMed

Artificial enzyme mimics have recently attracted considerable interest because they possess many advantages compared with natural enzymes, such as low cost of preparation and high stability. Herein, we present a novel fluorescent artificial enzyme-linked immunoassay strategy by utilizing Pd/C nanocatalyst as the enzyme mimic and bis-allyloxycarbonyl rhodamine 110 (BI-Rho 110) as the substrate, and the amplification procedure is based on the palladium-catalyzed Tsuji-Trost reaction. Pd/C nanocatalyst with the average size of 150 nm was prepared by the impregnation-reduction method, and high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) analyses reveal that Pd clusters with an average size of about 1 nm are dispersed uniformly on each carbon nanosphere's surface. Kinetic studies show that this reaction follows Michaelis-Menten kinetics and the fluorescence intensity is proportional to the concentration of Pd/C nanocatalyst under certain conditions. The turnover number of Pd/C nanocatalyst reaches up to 3.3 × 10(7) (h(-1)). The analytical performance of this system in detecting hCG shows that after a 24 h incubation the sensitivity limit can reach 0.1 ng/mL and the dynamic linear working range is 1-10 ng/mL. Our findings pave the way to use Pd-catalyzed reaction for design and development of novel analytical methods. PMID:24160777

Wang, Zhifei; Zheng, Shuang; Cai, Jin; Wang, Peng; Feng, Jie; Yang, Xia; Zhang, Liming; Ji, Min; Wu, Fugen; He, Nongyue; Wan, Neng

2013-12-01

71

Evaluation of an enzyme-linked immunoassay for the detection in serum of antibodies to Entamoeba histolytica  

Microsoft Academic Search

A commercially available enzyme-linked immunoassay (ELISA) was compared to an indirect hemagglutination test (IHA) for the detection of antibodies to Entamoeba histolytica on 225 patients' serums. All of the 107 serums that had IHA titers of <32 (interpreted as excluding the presence of invasive amebiasis) were ELISA negative. Sixty-three of 68 (93%) serums that had IHA titers of ?128 (interpreted

Jon E. Rosenblatt; Lynne M. Sloan; Jean E. Bestrom

1995-01-01

72

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

PubMed

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33?pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented. PMID:23785024

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

2014-06-01

73

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

USGS Publications Warehouse

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.

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

2001-01-01

74

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

SciTech Connect

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).

Marx, A.; Kroetz, E.; Hock, B. [Technische Univ. Muenchen, Freising (Germany). Dept. of Botany

1998-07-01

75

Evaluation of enzyme immunoassay for hepatitis B virus DNA based on anti-double-stranded DNA.  

PubMed Central

We have evaluated a new enzyme immunoassay technology to detect the products of PCR-based amplification that may be applicable to routine testing of hepatitis B virus (HBV) DNA. Two hundred eight serum samples were studied: 73 were basal samples and 135 were sequential serum samples from patients with chronic hepatitis, some of whom were being treated with alpha interferon. We compared the new detection method (PCR-DNA enzyme immunoassay [DEIA]) with dot blot hybridization performed without prior PCR amplification and with two other methods for detection of PCR products: agarose gel electrophoresis with ethidium bromide staining (PCR-EB) and dot blot (PCR-dot blot). For hepatitis B-antigen-positive basal samples, HBV DNA was detected in 70.4% by dot blot, 74.1% by PCR-EB, and 100% by PCR-DEIA and PCR-dot blot; for anti-hepatitis B e-antigen basal samples, HBV DNA was found in 10.5% by dot blot and PCR-EB and in 42.1% by PCR-DEIA and PCR-dot blot. Chi-square tests showed a strong association between dot blot and PCR-EB and between PCR-DEIA and PCR dot blot. Using PCR-dot blot as the reference, dot blot shows a 56.9% sensitivity and a 100% specificity, PCR-EB shows a 55.0% sensitivity and a 100% specificity, and PCR-DEIA shows a 95.4% sensitivity and a 97% specificity. We conclude that the technical advantages of the DEIA method and its high sensitivity and specificity may facilitate the use of PCR in routine testing for HBV DNA in clinical microbiology laboratories. PMID:7714201

Garcia, F; Garcia, F; Bernal, M C; Leyva, A; Piedrola, G; Maroto, M C

1995-01-01

76

Enzyme immuno assay for the detection of virus specific IgG and IgM antibody in patients with haemorrhagic fever with renal syndrome.  

PubMed

Consecutive serum samples collected from 235 patients with Haemorrhagic Fever with Renal Syndrome (HFRS), between two days and two years after onset of disease, have been analysed for the presence of IgG and IgM type of antibodies specific for Hanta-viruses. The sera were screened in parallel by a newly developed indirect Immuno Enzyme Assay (EIA) in parallel with Indirect Immunofluorescent Antibody Assay (IFA). In both tests the Hantaan virus strain 76-118 was used as the antigen. The EIA was much more sensitive than the IFA test for the detection of IgM type antibodies. With the indirect EIA IgM type antibodies against Hantaan virus 76-118 have been detected in HFRS patient's sera from the second day of illness indicating the usefulness of this test for the early serological diagnosis of this disease. PMID:2898929

Ivanov, A P; Tkachenko, E A; Petrov, V A; Pashkov, A J; Dzagurova, T K; Vladimirova, T P; Voronkova, G M; van der Groen, G

1988-01-01

77

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

PubMed

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method. PMID:20095621

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

2010-02-15

78

Taxane-specific monoclonal antibodies: measurement of taxol, baccatin III, and "total taxanes" in Taxus brevifolia extracts by enzyme immunoassay.  

PubMed

Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts. PMID:7561893

Grothaus, P G; Bignami, G S; O'Malley, S; Harada, K E; Byrnes, J B; Waller, D F; Raybould, T J; McGuire, M T; Alvarado, B

1995-07-01

79

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

PubMed

A substantial incidence of positive methadone screens for pain management urine specimens using a commercial enzyme immunoassay (EIA) was observed in the absence of a methadone prescription, with negative methadone confirmation by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS). Tapentadol was the only common prescription among the investigated specimens. Tapentadol or one of its three major metabolites was tested at various concentrations (100-200,000 ng/mL) against the DRI EIAs for methadone and methadone metabolite, to evaluate cross-reactivity. Ninety-seven authentic tapentadol urine specimens that produced false-positive methadone EIA results (cutoff = 130 ng/mL) were analyzed for methadone and tapentadol in compound-specific UPLC-MS-MS confirmation tests. Tapentadol, tapentadol glucuronide, tapentadol sulfate and N-desmethyltapentadol exhibited cross-reactivity with the methadone EIA at 6,500 (2.2%), 25,000 (0.6%), 3,000 (4.4%) and 20,000 ng/mL (0.9%), respectively. No cross-reactivity was observed with the methadone metabolite 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine EIA. All authentic urine specimens were confirmed to be negative for methadone, but positive for tapentadol and all monitored metabolites. Individual concentrations indicated that separate or combined urinary concentrations of tapentadol and its conjugates may produce false-positive methadone screens through cross-reactivity with the methadone immunoassay. The potential for false-positive results for methadone EIA screening of urine specimens associated with tapentadol prescriptions should be considered when interpreting results. PMID:22879537

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

2012-10-01

80

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

SciTech Connect

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.

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

1987-06-29

81

Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum.  

PubMed

A specific and sensitive immunoassay based on magnetic microbead separation for schistosomiasis japonica screening is presented in this article. So far as we know, this is the first time that magnetic microbead-based enzyme-linked immunoassay (MEIA) has been used for the determination of Schistosoma japonicum (Sj) antibody in human serum. Fluorescein isothiocyanate (FITC)-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that the MEIA method was more sensitive and more precise than traditional SEA-ELISA (enzyme-linked immunosorbent assay). In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect hemagglutination assay (IHA), dipstick dye immunoassay (DDIA), and MEIA. IHA and DDIA were then compared with MEIA, and a lower false negative rate (0.97%) was obtained. PMID:20470746

Liu, Zhenshi; Zhang, Li; Yang, Hai; Zhu, Yanhong; Jin, Wei; Song, Qian; Yang, Xiangliang

2010-09-15

82

Evaluation of Oral Fluid Enzyme Immunoassay for Confirmation of a Positive Rapid Human Immunodeficiency Virus Test Result?  

PubMed Central

The CDC recommends that a reactive rapid human immunodeficiency virus (HIV) test be confirmed with an approved supplemental test; the performance of an intermediate enzyme immunoassay (EIA) is optional. In support of this recommendation, it was found that of 1,431 reactive rapid HIV test results, 2 (0.1%) had false-negative oral fluid Western blot results and both had false-negative EIA results. PMID:19458205

Wesolowski, L. G.; Sanchez, T.; MacKellar, D. A.; Branson, B. M.; Ethridge, S. F.; Constantine, N.; Ketema, F.; Sullivan, P. S.

2009-01-01

83

Evaluation of oral fluid enzyme immunoassay for confirmation of a positive rapid human immunodeficiency virus test result.  

PubMed

The CDC recommends that a reactive rapid human immunodeficiency virus (HIV) test be confirmed with an approved supplemental test; the performance of an intermediate enzyme immunoassay (EIA) is optional. In support of this recommendation, it was found that of 1,431 reactive rapid HIV test results, 2 (0.1%) had false-negative oral fluid Western blot results and both had false-negative EIA results. PMID:19458205

Wesolowski, L G; Sanchez, T; MacKellar, D A; Branson, B M; Ethridge, S F; Constantine, N; Ketema, F; Sullivan, P S

2009-07-01

84

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA)  

Microsoft Academic Search

A protein-capture fluorescence enzyme immunoassay (FEIA) was developed using monoclonal antibodies (mAbs) against recombinant hepatitis C virus (HCV) core protein. Four hybridoma cell lines (5E3, 5F11, 515S, 1080S) were established and characterized. These monoclonal antibodies (mAbs) each had IgG1 and IgG2 isotypes, and recognized major B cell epitopes within the immunodominant nucleoprotein amino terminal subregion. Using mAb 5F11 as the

Tomiko Kashiwakuma; Akira Hasegawa; Tadahiro Kajita; Atsumi Takata; Hiroyuki Mori; Yohsuke Ohta; Eiji Tanaka; Kendo Kiyosawa; Takeshi Tanaka; Satoshi Tanaka; Nobu Hattori; Michinori Kohara

1996-01-01

85

Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load  

Microsoft Academic Search

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore\\/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc\\/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simulta-

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

2002-01-01

86

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720  

Microsoft Academic Search

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies

Norimasa Matsumoto; Takeyuki Kohno; Shuji Uchida; Takaaki Shimizu; Haruko Kusumoto; Ryoji Hirose; Kazuo Yanada; Wasako Kurio; Shoji Yamaguchi; Kazuhito Watabe; Tetsuro Fujita

2006-01-01

87

Evaluation of influenza virus detection by direct enzyme immunoassay (EIA) and conventional methods in asthmatic patients.  

PubMed

Throat gargle specimens of fifty-seven acute asthmatic patients (age range 18-40 years) were collected for the study. Thirty-four patients were found influenza virus positive in acute asthma cases. Influenza virus was isolated by conventional culture method on MDCK cell-line and by enzyme immunoassay test (EIA). The EIA negative specimens were retested after virus amplification on MDCK cell-line. Virus shedding and virus surface receptors assay was carried out to determine influenza virus titre. Airway functions were measured by spirometry. A good relationship was observed between the degree of airflow limitation and presence of influenza virus infection (p < 0.001; r = 0.85). A comparable difference in % FEV1 was observed in relation to the symptoms. The patients with greater viral antigen load had lower % FEV1. Two specimens, which were EIA negative, turned out to be positive after amplification on MDCK cell-line. The sensitivity was 98% and specificity was 100%. It was concluded that EIA method is a useful diagnostic tool as it detects influenza viral antigen quickly as compared to conventional methods. PMID:12206034

Khanna, M; Kumar, P; Chugh, L; Prasad, A K; Chhabra, S K

2001-09-01

88

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.  

PubMed

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. PMID:24769049

Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

2014-08-15

89

Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay.  

PubMed

A chemiluminescence enzyme immunoassay (CLEIA) based on the HRP-luminol-H?O? chemiluminescence system for highly sensitive detection of enrofloxacin (ENR) was proposed in this study. Key factors that affect the precision and accuracy for the determination of ENR residues were optimised. Under the optimal conditions, the proposed method showed an excellent performance. The linearity range for method developed for determination of ENR was 0.35-1.0 ng/mL with a correlation coefficient greater than 0.994. The limit of detection was 0.03 ng/mL and the relative standard deviations (RSDs) were less than 9.4% and 13.0% for intra-day and inter-day assays. The proposed method was satisfactorily applied to determine ENR in milk, eggs, and honey samples at three spiked levels (0.4, 0.7, and 1.0 ng/mL) and the recoveries ranged from 92.4% to 104.2% for milk, 93.8% to 103.2% for eggs and 94.1% to 105.0% for honey, respectively. Compared the results of CLEIA with those of ELISA and HPLC, the advantages of the CLEIA were further confirmed. Moreover, one 96-well microtiter plate coated with anti-ENR can be used to detect multiple samples at the same time, which indicated that the CLEIA using HRP-luminol-H?O? system was a sensitive, high throughput and real-time method for ENR residues analysis. PMID:24295678

Yu, Fei; Yu, Songcheng; Yu, Lanlan; Li, Yanqiang; Wu, Yongjun; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B

2014-04-15

90

Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells.  

PubMed

Complete parasite development was obtained in differentiated human enterocytic HCT-8 cells infected at confluence with Cryptosporidium parvum sporozoites, and evaluated in a quantitative enzyme immunoassay. Forty-eight hours after infection, a linear correlation was found between optical density values and the number of parasites determined in an immunofluorescent assay. Sinefungin exerted an inhibitory effect when added within 4 h after sporozoite addition to HCT-8 cultures (MIC50 = 38 mumol L-1), while the inhibitory effects of paromomycin and pentamidine dimethanesulfonate were also easily detected (MIC50 = 0.87 mumol L-1 and 0.27 mumol L-1, respectively). Except for high pentamidine dimethanesulfonate concentrations, no alteration in optical microscopy morphology or trypan blue exclusion of HCT-8 cells was observed in the presence of anticryptosporidial agents, which suggests that they were primarily active against developing parasites. Data suggest that EIA detection of C. parvum development in sporozoite-infected HCT-8 cells provides an accurate and convenient model for in vitro evaluation of parasite infectivity, growth and response to anticryptosporidial agents. PMID:10404264

Gargala, G; Delaunay, A; Favennec, L; Brasseur, P; Ballet, J J

1999-05-01

91

Measuring multiple hormones from a single water sample using enzyme immunoassays.  

PubMed

Many aquatic species, such as teleosts, release into the water and detect multiple bioactive substances to assist in schooling, migration, alarm reactions, and to stimulate behavioral and physiological responses during reproduction and in parent-offspring interactions. Understanding the complex relationship between hormones, behavior and their function in communication requires the simultaneous examination of multiple circulating hormones. However, repeated blood sampling within a short time period is not possible in smaller animals without impacting the very behaviors under investigation. The non-invasive technique of collecting and measuring hormone values in holding water using either radioimmunoassay (RIA) or enzyme immunoassay (EIA) is becoming widely used in teleost research. Commercial assay kits in particular enable rapid and reliable data generation, yet their assay buffers are often specific and potentially incompatible with each other, which can hinder measuring multiple hormones from the same sample. We present here the validation and application of a "nested" elution technique we developed that allows for repeated sampling of multiple reproductive hormones - testosterone (T), 17beta-estradiol (E2), progesterone (P), prostaglandin F(2 alpha) (PGF) and 11-ketotestosterone (11KT) - from individual samples of animal holding water by using commercial EIA systems. Our results show that when using appropriate controls to account for possible technical and biological confounds, this technique provides a powerful new tool for research in aquatic endocrinology and physiology. PMID:19607832

Kidd, Celeste E; Kidd, Michael R; Hofmann, Hans A

2010-01-15

92

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

PubMed

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

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

93

Comparison of two commercially available enzyme immunoassays for detection of Clostridium difficile in stool specimens.  

PubMed Central

Clostridium difficile is the cause of most cases of pseudomembranous colitis, the most severe form of antibiotic-associated diarrhea. Rapid diagnosis guides both the treatment and the control of nosocomial spread of infection. Two enzyme immunoassay (EIA) kits developed for the rapid detection of C. difficile toxin A in fecal specimens, Premier (Meridian Diagnostics, Cincinnati, Ohio) and Tox-A test (TechLab, Virginia Polytechnic Institute Research Park, Blacksburg), were evaluated by using 410 fecal specimens. Seventy-six specimens were positive for C. difficile toxin B by the cytotoxin assay (prevalence rate, 19%). The Meridian EIA was positive for 71 of the 76 samples, yielding a sensitivity of 93%. The TechLab EIA detected 75 of the 76 positive samples, yielding a sensitivity of 99%. The Meridian and TechLab EIAs had specificities of 100 and 93%, respectively. These data indicate that both EIAs are suitable alternatives to the cytotoxin assay in routine diagnostic laboratories. However, confirmation of TechLab EIA-positive test results by the cytotoxin assay remains necessary. PMID:8126205

Altaie, S S; Meyer, P; Dryja, D

1994-01-01

94

Comparison of salivary and serum enzyme immunoassays for the diagnosis of Helicobacter pylori infection  

PubMed Central

Infection with Helicobacter pylori has been established as an important risk factor for the development of peptic ulcer disease, gastritis and gastric cancer. The diagnosis of H pylori infection can be established by invasive or noninvasive techniques. Two noninvasive enzyme immunoassays (EIAs) for antibody detection – HeliSal and Pylori Stat – were compared with histology. Both assays detect immunoglobulin (Ig) G directed against purified H pylori antigen. The test populations consisted of 104 consecutive patients scheduled for upper gastrointestinal endoscopy. Of these patients, 97 (93%) had symptoms compatible with peptic ulcer disease. Saliva and serum were collected simultaneously at the time of endoscopy. Salivary EIA had a sensitivity of 66%, specificity of 67%, positive predictive value of 67% and negative predictive value of 66% compared with the serum EIA, where the results were 98%, 48%, 64% and 96%, respectively. Although the salivary EIA is an appealing noninvasive test, it was not a sensitive and specific assay. The serum EIA also lacked specificity, but was highly sensitive with a good negative predictive value. Although a negative serum EIA rules out H pylori infection, a positive result must be interpreted in the clinical context and confirmed with a more specific measure. PMID:22346548

Embil, John M; Choudhri, Shurjeel H; Smart, Gerry; Aldor, Thomas; Pettigrew, Norman M; Grahame, Gordon R; Dawood, Magdy R; Bernstein, Charles N

1998-01-01

95

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

PubMed Central

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

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

2014-01-01

96

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

PubMed

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

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

2011-12-01

97

Dot enzyme immunoassay for visual detection of peste-des-petits-ruminants virus antigen from infected caprine tissues.  

PubMed Central

An enzyme-linked immunosorbent microassay using nitrocellulose paper as the solid-phase support was developed for the detection of peste-des-petits-ruminants virus antigens in infected caprine tissue homogenates. Dots of tissue homogenates were applied to nitrocellulose papers, and any unreacted sites were blocked with 5% skim milk powder in triethanolamine-buffered saline. After incubation of the papers in tissue culture supernatant monoclonal antibody against the peste-des-petits-ruminants virus, the antigen-antibody reaction was detected with peroxidase-conjugated anti-mouse immunoglobulin G and the enzyme substrate 4-chloro-1-naphthol. Positive results were visualized as blue dots. Results of the dot enzyme immunoassay compared favorably with those of the standard enzyme-linked immunosorbent assay. Incorporation of Nonidet P-40 in the washing solution did not improve the sensitivity of the dot enzyme immunoassay, and pretreatment of homogenates with Nonidet P-40 before application to the nitrocellulose paper inhibited the binding of the antigen to the paper and reduced the intensity of the color development. Images PMID:2674200

Obi, T U; Ojeh, C K

1989-01-01

98

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

PubMed

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

Adel Ahmed, Heba; Azzazy, Hassan M E

2013-11-15

99

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.  

PubMed

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation. PMID:16491341

Roda, A; Mirasoli, M; Guardigli, M; Michelini, E; Simoni, P; Magliulo, M

2006-03-01

100

Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients  

PubMed Central

We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml?1 limit of detection and thus ?100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml?1 limit of detection was observed) on a minimum of 30 ?l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients. PMID:14693526

Azoulay, Stephane; Nevers, Marie-Claire; Creminon, Christophe; Heripret, Laurence; Durant, Jacques; Dellamonica, Pierre; Grassi, Jacques; Guedj, Roger; Duval, Daniele

2004-01-01

101

Evaluation of the fully automated Cobas Core enzyme immunoassay for the quantitation of antibodies against hepatitis B virus surface antigen.  

PubMed

The Roche Cobas Core automated enzyme immunoassay of antibodies against hepatitis B virus surface antigen (anti-HBs) was evaluated using a protocol complying with the Société Française de Biologie Clinique guidelines. Results showed good precision (CV below 6 and 9% for intra- and inter-assay precision) and accuracy (according to the Paul-Ehrlich-Institute standard); the detection limit of the test was 2 IU/l. The manufacturer's specifications were confirmed and a satisfactory correlation (r = 0.901) was found with an automated method (IMx, Abbott) used for comparison. PMID:8704055

Doche, C; Thomé, M; Dimet, I; Bienvenu, J

1996-04-01

102

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

PubMed Central

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

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

2003-01-01

103

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

SciTech Connect

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.

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

2011-09-09

104

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

PubMed Central

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

DeLuca, Jane

2007-01-01

105

Performance of Commercially Available Enzyme Immunoassays for Detection of Antibodies against Herpes Simplex Virus Type 2 in African Populations  

PubMed Central

Data are accumulating on the performance of enzyme immunoassays (EIAs) for the detection of herpes simplex virus type 2 (HSV-2) infection in North America and Europe, but little is known about their performance in other populations. Nine test kits were evaluated with 330 serum samples from sub-Saharan Africa. The tests were first compared to the monoclonal antibody (MAb) EIA (Central Public Health Laboratory, London, United Kingdom). Samples that gave discordant results in the MAb EIA and in the three tests that performed best compared to the MAb EIA were tested by Western blotting (University of Washington, Seattle). A random sample of concordant samples was also tested, and the sensitivities and specificities of the different tests were calculated, taking into account this sampling strategy. The sensitivities of the tests ranged from 86 to 100%; the specificities ranged from 47 to 99%. The tests that performed best were the Gull Premier EIA (sensitivity, 86.3%; specificity, 97.6%) and the Kalon Biological (sensitivity, 92.3%; specificity, 97.7%) and Biokit (sensitivity, 86.7%; specificity, 92.6%) tests. It cannot be assumed that enzyme immunoassays for the detection of HSV-2 infection that perform well in industrialized countries will perform equally well in other populations. PMID:15243045

Van Dyck, Eddy; Buve, Anne; Weiss, Helen A.; Glynn, Judith R.; Brown, David W. G.; De Deken, Benedicte; Parry, John; Hayes, Richard J.

2004-01-01

106

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.  

PubMed

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

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

107

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

PubMed Central

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

Takehara, Shizuka; Takahashi, Masaharu

2013-01-01

108

Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.  

PubMed

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. PMID:24723253

Yang, Tingzhen; Vdovenko, Marina; Jin, Xue; Sakharov, Ivan Yu; Zhao, Shulin

2014-07-01

109

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection  

PubMed Central

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a “gold standard” for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The test's positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection. PMID:15242956

Attallah, Abdelfattah M.; Ismail, Hisham; Ibrahim, Gellan G.; Abdel-Raouf, Mohamed; El-Waseef, Ahmed M.; Abdel-Wahab, Mohamed

2004-01-01

110

Evaluation of a New Dot Blot Enzyme Immunoassay (Directigen Flu A+B) for Simultaneous and Differential Detection of Influenza A and B Virus Antigens from Respiratory Samples  

Microsoft Academic Search

We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples

Jordi Reina; Emma Padilla; Fermin Alonso; Enrique Ruiz de Gopegui; Maria Munar; Margarita Mari

2002-01-01

111

Evaluation of a New Dot Blot Enzyme Immunoassay (Directigen Flu AB) for Simultaneous and Differential Detection of Influenza A and B Virus Antigens from Respiratory Samples  

Microsoft Academic Search

We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples

Jordi Reina; Emma Padilla; Fermin Alonso; Enrique Ruiz de Gopegui; Maria Munar

112

Application of a Polymerase Chain Reaction Enzyme Immunoassay in Peripheral Whole Blood and Serum Specimens for Diagnosis of Acute Human Brucellosis  

Microsoft Academic Search

A simple polymerase chain reaction-enzyme immunoassay (PCR-EIA) was employed for the rapid laboratory diagnosis of human brucellosis directly from peripheral blood. Whole blood and serum specimens were collected from 243 patients with acute brucellosis as determined by blood culture, serological tests, and the patients’ clinical characteristics and from a control group of 50 healthy individuals. Diagnosis of brucellosis was established

G. Vrioni; C. Gartzonika; A. Kostoula; C. Boboyianni; C. Papadopoulou; S. Levidiotou

2004-01-01

113

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

USGS Publications Warehouse

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.

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

114

Evaluation of the PATHFAST Chemiluminescent Enzyme Immunoassay for Measuring Progesterone in Whole Blood and Serum of Mares  

PubMed Central

Evaluation of a new chemiluminescent enzyme immunoassay, the PATHFAST assay system (PATHFAST), for measurement of circulating progesterone in mares was performed. Five mares at the mid-luteal stage were administrated a single i.m. injection of prostaglandin F2? analog (PGF2?; cloprostenol 250 ?g/ml), and then blood samples were collected from the jugular vein at 0, 15, 30 and 45 min, at one-hour intervals until 24 and at 48 hr via a catheter in the jugular vein. To monitor the physiological changes in circulating progesterone in mares after induced luteolysis, concentrations of progesterone in whole blood and serum samples were measured by PATHFAST. In addition, concentrations of progesterone in serum samples measured by PATHFAST were compared with those measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA). Using PATHFAST, the serum concentrations of progesterone in mares correlated highly with those of whole blood samples (r=0.9672, n=88). The serum concentrations of progesterone as measured by PATHFAST correlated well with RIA (r=0.9654, n=88) and EIA (r=0.9323, n=112). An abrupt decline in circulating progesterone in whole blood samples was observed within 2 hr (50%), followed by a gradual decline until 48 hr later. The results for progesterone in whole blood samples correlated highly with those in serum samples, and the declining pattern paralleled that of the serum samples. These results demonstrated that PATHFAST is useful in the equine clinic as an accurate diagnostic tool for rapid assay of progesterone within 26 min, using unextracted whole blood. PMID:24834001

TOISHI, Yuko; TSUNODA, Nobuo; TAGAMI, Masaaki; HASHIMOTO, Hiromitsu; KATO, Fumiki; SUZUKI, Tsukasa; NAGAOKA, Kentaro; Watanabe, Gen; TOKUYAMA, Shota; OKUDA, Kiyoshi; TAYA, Kazuyoshi

2013-01-01

115

Evaluation of an IgM-specific indirect enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infection: influence of IgM rheumatoid factor on test results with field sera.  

PubMed

A commercially available indirect enzyme-linked immunosorbent assay for measuring bovine respiratory syncytial virus (BRSV)-specific IgG was adapted to measure virus-specific IgM. Using this assay, the development of rapid IgM responses in experimentally infected calves was observed 7-9 days postinfection, with peak absorbance values ranging from 1.698 to 2.873. When absorbance values were expressed as a percentage of a positive reference serum, a positive/negative threshold of 22% was determined by testing serum samples from 59 healthy 3-5-month-old calves. Acute and convalescent serum samples collected from 151 calves during 38 outbreaks of respiratory disease were tested, and 130 sera were positive. To determine the number of false-positive results due to the presence of IgM rheumatoid factor, a method for depleting serum IgG by pretreatment of sera with a suspension of protein-G-agarose was developed. All sera that initially tested IgM positive were retested following depletion of serum IgG. False-positive IgM reactions were detected in 23 sera (17.7%). Specific IgM responses were confirmed in 107 sera from 84 calves. Evidence of BRSV infection was detected in 34 of 38 outbreaks. In contrast, seroconversion was detected in 69 calves from 24 outbreaks, confirming the diagnostic potential of the IgM assay. Overall correlation between IgM and seroconversion results was 74.2%. Intra- and interassay reproducibility were 12.50% and 17.48%, respectively (mean coefficients of variation). PMID:9786520

Graham, D A; Mawhinney, K A; Elvander, M; Adair, B M; Merza, M

1998-10-01

116

Immunoassays in Environmental Analytical Chemistry  

Microsoft Academic Search

A review is presented which summarizes the recent developments of immunoassays in environmental analytical chemistry.The basic principle of the method and the following steps in the development of an immunoassay procedure are discussed in detail: Synthesis of the immunogen, immunization procedure, synthesis of the labelled antigen (tracer), advantages and drawbacks of radioimmunoassay, fluoroimmunoassay and enzyme immunoassay. A special emphasis is

Margit Schwalbe-fehl

1986-01-01

117

Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load  

PubMed Central

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 103 U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 × 102 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients. PMID:11825954

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

2002-01-01

118

A paper-based surface-enhanced resonance Raman spectroscopic (SERRS) immunoassay using magnetic separation and enzyme-catalyzed reaction.  

PubMed

In this study, a novel paper-based SERRS immunoassay based on magnetic separation and alkaline phosphatase (ALP) enzyme catalyzed hydrolysis reaction was developed. By using modified antibodies conjugated to magnetic beads, capture of mouse IgG followed by addition of ALP-labeled antibodies would form a sandwich-like immunoconjugate. After magnetic separation, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), a low SERRS active compound, was added as the substrate for ALP to generate a high SERRS response. Detection was conducted on a silver colloid/PVP/filter paper SERS substrate by spotting a pre-aggregated silver colloid sol onto polyvinyl pyrrolidone (PVP) modified filter paper using a semi-automatic TLC sample applicator. The optimization of the highly SERS active paper-based substrate, dynamic hydrolysis process of BCIP, quantitative detection of IgG, and selectivity of the assay was studied in detail. By taking advantage of magnetic separation in order to decrease the background interference, the selective enzyme reaction involved in producing a highly SERRS active product could detect mouse IgG from 1 to 500 ng mL(-1) with a LOD of 0.33 ng mL(-1). PMID:23486763

Chen, Yuanyuan; Cheng, Hanwen; Tram, Kha; Zhang, Shengfeng; Zhao, Yanhua; Han, Liyang; Chen, Zengping; Huan, Shuangyan

2013-05-01

119

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

PubMed Central

Anti-nuclear antibodies (ANA) have traditionally been evaluated using indirect fluorescence assays (IFA) with HEp-2 cells. Quantitative immunoassays (EIA) have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique patients were compared. The majority of the results were the same between the two methods (n = 325, 84%); however, 8% (n = 30) yielded equivocal results (equivocal-negative and equivocal-positive) and 8% (n = 31) yielded divergent results (positive-negative). The results showed fairly good agreement, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14–0.46), which decreased to 0.23 (95% CI = 0.06–0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test. PMID:24592328

Baronaite, Renata; Engelhart, Merete; M?rk Hansen, Troels; Thamsborg, Gorm; Slott Jensen, Hanne; Stender, Steen; Szecsi, Pal Bela

2014-01-01

120

Double layer enzyme modified carbon nanotubes as label for sandwich-type immunoassay of tumor markers  

Microsoft Academic Search

An immunosensor was prepared for the determination of carcinoembryonic antigen (CEA). It is based on the use of multiwalled\\u000a carbon nanotubes (MWCNTs) along with horseradish peroxidase-labeled antibody. The enzyme was assembled onto MWCNTs templates\\u000a using the layer-by-layer technique and then conjugated to carcinoembryonic secondary antibodies (Ab2) as the enzyme label. The resulting assembly results in a largely amplified sensitivity. The

Min Liang; Ruo Yuan; Yaqin Chai; Ligen Min; Zhongju Song

2011-01-01

121

Validation of an Enzyme Immunoassay for Detection and Semiquantification of Cannabinoids in Oral Fluid  

PubMed Central

BACKGROUND Oral fluid (OF) is gaining prominence as an alternative matrix for monitoring drugs of abuse in the workplace, criminal justice, and driving under the influence of drugs programs. It is important to characterize assay performance and limitations of screening techniques for ?9-tetrahydrocannabinol (THC) in OF. METHODS We collected OF specimens by use of the Quantisal™ OF collection device from 13 daily cannabis users after controlled oral cannabinoid administration. All specimens were tested with the Immunalysis Sweat/OF THC Direct ELISA and confirmed by 2-dimensional GC-MS. RESULTS The limit of detection was <1 µg/L THC equivalent, and the assay demonstrated linearity from 1 to 50 µg/L, with semiquantification to 200 µg/L. Intraplate imprecision (n = 7) ranged from 2.9% to 7.7% CV, and interplate imprecision (n = 20) was 3.0%–9.1%. Cross-reactivities at 4 µg/L were as follows: 11-hydroxy-THC, 198%; ?8-tetrahydrocannabinol (?8-THC), 128%; 11-nor-9-carboxy-THC (THCCOOH), 121%; THC (target), 98%; cannabinol, 87%; THCCOOH-glucuronide, 11%; THC-glucuronide, 10%; and cannabidiol, 2.4%. Of 499 tested OF specimens, 52 confirmed positive (THC 2.0–290 µg/L), with 100% diagnostic sensitivity at the proposed Substance Abuse and Mental Health Services Administration screening cutoff of 4 µg/L cannabinoids and GC-MS cutoff of 2 µg/L THC. Forty-seven specimens screened positive but were not confirmed by 2D-GC-MS, yielding 89.5% diagnostic specificity and 90.6% diagnostic efficiency. Thirty-one of 47 unconfirmed immunoassay positive specimens were from 1 individual and contained >400 ng/L THCCOOH, potentially contributing to cross-reactivity. CONCLUSIONS The Immunalysis Sweat/OF THC Direct ELISA is an effective screening procedure for detecting cannabinoids in OF. PMID:20360126

Schwope, David M.; Milman, Garry; Huestis, Marilyn A.

2011-01-01

122

Relevance of estrogen and progesterone receptors enzyme immunoassay in malignant, benign and surrounding normal thyroid tissue.  

PubMed

Several authors have demonstrated the presence of estrogen receptors (ER) and progesterone receptors (PR) in thyroid tissue, generally using dextran coated charcoal method (DCCA). The aim of the study was to measure ER and PR in thyroid specimens using an immunoenzymatic method, and to evaluate the meaning of different prevalence of ER and PR in malignant and benign thyroid disease, as compared with normal thyroid tissue. We have measured ER and PR in thyroid tissue from 28 benign and 20 malignant thyroid lesions, and in 38 samples of surrounding normal thyroid tissues. The sensitivity of ER-EIA and PR-EIA was 1.0 and 1.5 fmol/mg protein, respectively. In thyroid carcinoma the frequency of ER positivity (ER+) was 7/20 (35%); it was significantly higher in the surrounding normal tissue (15/20; 71%) (p = 0.03). In benign thyroid disease, the prevalence of ER+ was 11/28 (39%) and in the surrounding normal tissue it was 11/18 (61%) (p = not significant). PR+ was detected in 7/20 (35%) thyroid cancers and in 15/28 (53%) benign lesions without significant difference with the frequency detected in the surrounding normal tissues. ER and PR concentrations (mean +/- SD) in thyroid cancer was 2.2 +/- 2.2 and 2.2 +/- 2.9 respectively, similarly to that detected in benign thyroid disease and in normal tissue. The simultaneous presence of ER and PR (ER+PR+) was also evaluated. We have found that the frequency of ER+ PR+ was significantly higher in benign lesions (8/28; 28.6%) as compared with malignant samples (1/20; 5%) (p < 0.05); the frequency of ER+PR+ was significantly higher in normal tissue surrounding the malignant lesions (9/20; 45) (p = 0.003). Our data indicate i) EIA method is appropriate to detect ER and PR in thyroid tissue. ii) The frequency of ER+ and ER+PR+ specimens is significantly higher in normal thyroid tissue than in pathologic tissues. This indicates that ER and PR immunoassays may be useful tools to evaluate the normal biological activity of thyroid cells. PMID:8743281

Bonacci, R; Pinchera, A; Fierabracci, P; Gigliotti, A; Grasso, L; Giani, C

1996-03-01

123

Comparison of the Directigen Flu AB Membrane Enzyme Immunoassay with Viral Culture for Rapid Detection of Influenza A and B Viruses in Respiratory Specimens  

Microsoft Academic Search

The performance of a commercially available, rapid membrane enzyme immunoassay for influenza A and B virus detection was compared to that of viral culture in 4,092 respiratory specimens collected from patients presenting with respiratory symptoms during the 2002-2003 influenza season. The test's overall sensitivity was 43.83%, lower than previously reported but similar for detection of both influenza A and B

Andreea C. Cazacu; Sooyoung E. Chung; Jewel Greer; Gail J. Demmler

124

Evaluation of a new enzyme immunoassay for hepatitis C virus (HCV) core antigen with clinical sensitivity approximating that of genomic amplification of HCV RNA  

Microsoft Academic Search

The aim of this study was to analyze the clinical performance of a new enzyme immunoassay (EIA) for hepatitis C virus (HCV) core antigen in comparison with the reverse transcription polymerase chain reaction (RT-PCR). A total of 310 patients with acute or chronic hepatitis C, and 132 HCV-negative controls were studied. Chemiluminescence EIA with monoclonal anti-HCV core antigen was used,

Eiji Tanaka; Chiharu Ohue; Katsumi Aoyagi; Kenjiro Yamaguchi; Shintaro Yagi; Kendo Kiyosawa; Harvey J. Alter

2000-01-01

125

Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing  

Microsoft Academic Search

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphi- lis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results

V. W. HALLING; M. F. JONES; J. E. BESTROM; A. D. WOLD; J. E. ROSENBLATT; T. F. SMITH; F. R. COCKERILL

1999-01-01

126

Noninvasive monitoring of fecal cortisol metabolites in the eastern chipmunk (Tamias striatus): validation and comparison of two enzyme immunoassays.  

PubMed

Monitoring fecal glucocorticoid metabolites in wild animals, using enzyme immunoassays, enables the study of endocrinological patterns relevant to ecology and evolution. While some researchers use antibodies against the parent hormone (which is typically absent from fecal samples), others advocate the use of antibodies designed to detect glucocorticoid metabolites. We validated two assays to monitor fecal cortisol metabolites in the eastern chipmunk (Tamias striatus). We compared an antibody produced against cortisol and one produced against 5?-pregnane-3?, 11?, 21-triol-20-one using a radiometabolism study and an injection with adrenocorticotropic hormone (ACTH). Most cortisol metabolites were excreted in the urine (?83%). Peak excretion in the feces occurred 8 h after injection. Both assays detected an increase in fecal cortisol metabolite levels after injection of ACTH. Males, but not females, exhibited a circadian variation in metabolite levels. The sexes did not exhibit any difference over the time course and route of excretion or the relative increase in fecal cortisol metabolite levels after ACTH injection. The cortisol assay displayed higher reactivity to ACTH injection relative to baseline than did the metabolite assay. While both antibodies gave comparable results, the cortisol antibody was more sensitive to changes in plasma cortisol levels in eastern chipmunks. PMID:22418710

Montiglio, Pierre-Olivier; Pelletier, Fanie; Palme, Rupert; Garant, Dany; Réale, Denis; Boonstra, Rudy

2012-01-01

127

Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle, buffaloes, and goats.  

PubMed

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 ?L of fresh plasma (for free cortisol) and also with 20 ?L of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 ?L were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 ?L, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals. PMID:23816261

Yadav, R; Mohan, K; Kumar, V; Sarkar, M; Nitu, K; Meyer, H H D; Prakash, B S

2013-08-01

128

Comparison of four enzyme immunoassays for detection of human T-cell lymphotropic virus type 2 antibodies.  

PubMed

Four licensed enzyme immunoassay (EIA) kits for the measurement of antibody to human T-cell lymphotropic virus (HTLV) type 1, one from Organon Teknika Corp. (OTC), one from Cambridge Biotech Corp. (CBC), and two from Abbott Laboratories (the 1993 modification [Abb 93] and the 2.0 version licensed in 1995 [Abb 95]), were evaluated for sensitivity and specificity in the detection of HTLV type 2 antibody, and the results were compared with those previously obtained with earlier kit versions. The CBC, Abb 95, Abb 93, and OTC kits had sensitivities of 99.7, 97.6, 96.8, and 96.2%, respectively, compared with sensitivities of 89.1 and 60% for the Abbott and CBC (previously DuPont) kits, respectively, licensed in 1988. Thus, the abilities of commercial kits to detect HTLV antibody have improved. The relative specificities of the CBC, Abb 95, Abb 93, and OTC kits with negative blood donor specimens that had been reactive with the 1988 CBC EIA kit were 92.9, 64.5, 78.8, and 62.6%, respectively. Compared with those of the 1988 versions, the specificity of the Abbott EIA has decreased and the specificity of the CBC kit has been significantly improved. PMID:8748309

Gallo, D; Yeh, E T; Moore, E S; Hanson, C V

1996-01-01

129

Evaluation of the modified Visuwell Strep-A enzyme immunoassay for detection of group-A Streptococcus from throat swabs.  

PubMed

The modified Visuwell Strep-A enzyme immunoassay (EIA) was compared with culture for detection of group-A Streptococcus from throat swabs. Throat swabs in modified Stuarts medium obtained after culture at two institutions were tested in Visuwell. Cumulative results were n = 417, sensitivity 87.8%, specificity 89.9% predictive value positive (PVP) 67.9%, predictive value negative (PVN) 96.8%, and accuracy 89.5%. At another site, swabs were delivered to the laboratory without transport medium, cultured, and subsequently tested by Visuwell (n = 202, sensitivity 79.6%, specificity 84.5%, PVP 65.2%, PVN 91.9%, accuracy 83.2%). When 1+ culture-positive specimens were considered negative, the sensitivity and PVN increased from 79.6% to 90.2% and 91.9% to 97.1% respectively. Overall performance of the modified Visuwell was comparable with that of the initial assay for throat swabs transported with or without modified Stuarts medium. Cross reaction with organisms other than group-A Streptococcus normally found in the oropharynx was negligible in Visuwell and the limit of detection of group-A Streptococcus was 5 x 10(4) colony-forming units. PMID:1889180

Drulak, M; Bartholomew, W; LaScolea, L; Amsterdam, D; Gunnersen, N; Yong, J; Fijalkowski, C; Winston, S

1991-01-01

130

Detection of Tritrichomonas foetus by PCR and DNA Enzyme Immunoassay Based on rRNA Gene Unit Sequences  

PubMed Central

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

Felleisen, Richard S. J.; Lambelet, Natacha; Bachmann, Philipp; Nicolet, Jacques; Muller, Norbert; Gottstein, Bruno

1998-01-01

131

Serum placental-like alkaline phosphatase (PLAP): a novel combined enzyme linked immunoassay for monitoring ovarian cancer.  

PubMed Central

A new combined enzyme linked immunoassay (ELISA) was developed to measure both serum placental-like alkaline phosphatase (PLAP) activity (PLAPA) and concentration (PLAPC) in the same microtitre plate using an Imperial Cancer Research Fund monoclonal antibody, designated H17E2. PLAP A and PLAP C were determined together with an existing marker, CA125 in 397 serial samples from 87 patients with epithelial ovarian cancer. Retrospective assessment showed the sensitivity to increase from 73% with CA125 alone, to 88% using CA125 and PLAP A, and to 93% with all three markers in 261 samples from the patients with known active disease at the time of sampling. When the results for all 397 samples were included in the analysis, however, the specificity, sensitivity, accuracy and predictive powers of this monoclonal antibody were not sufficiently high to assist in the prospective follow up of patients with ovarian cancer. This was due to a significant number of false positive and false negative results. Our data indicate that PLAP A or PLAP C estimation with H17E2 may, therefore, only be of value in the management of those patients with known active disease who are already known to be "marker positive" for this antigen. PMID:2921344

Fisken, J; Leonard, R C; Shaw, G; Bowman, A; Roulston, J E

1989-01-01

132

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli  

SciTech Connect

The authors have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 ..mu..g/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25/sup 0/C in the enzyme thermistor unit. Thus, immediate assay start up was possible.

Birnbaum, S.; Buelow, L.; Hardy, K.; Danielsson, B.; Mosbach, K.

1986-10-01

133

Double antibody sandwich enzyme linked immunoassay and rapid Immunoswab assay for detection of gliadin in food  

Microsoft Academic Search

Celiac (gluten intolerant) sprue is a life-long threatening disease. The only effective treatment thus far is to maintain a gluten-free diet. Sensitive and reliable double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and Immunoswab assay were developed by using chicken egg yolk anti-gliadin immunoglobulin Y as capturing antibody and monoclonal anti-gliadin IgG (HYB-314) antibody as detecting antibody for the detection of

Hoon H. Sunwoo; Naiyana Gujral; Susan Lutz; Mavanur Suresh

2011-01-01

134

Double antibody sandwich enzyme linked immunoassay and rapid Immunoswab assay for detection of gliadin in food  

Microsoft Academic Search

Celiac (gluten intolerant) sprue is a life-long threatening disease. The only effective treatment thus far is to maintain a gluten-free diet. Sensitive and reliable double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and Immunoswab assay were developed by using chicken egg yolk anti-gliadin immunoglobulin Y as capturing antibody and monoclonal anti-gliadin IgG (HYB-314) antibody as detecting antibody for the detection of

Hoon H. Sunwoo; Naiyana Gujral; Susan Lutz; Mavanur Suresh

2012-01-01

135

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

SciTech Connect

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.

Thurman, E.M.; Aga, D.S.; Zimmerman, L.R.; Goolsby, D.A. [Geological Survey, Lawrence, KS (United States)

1996-10-01

136

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

PubMed

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting aflatoxin M1 (AFM1) was developed. To improve the sensitivity of the assay, a mixture of 3-(10'-phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used to enhance peroxidase-induced CL. The concentrations of the coating anti-AFM1 antibody and the conjugate of AFB1 with horseradish peroxidase the conditions of the chemiluminescent assay were varied to optimise the condition of the chemiluminescent assay. The lower detection limit values and dynamic working range of CL-ELISA of AFM1 were 0.001 ng mL(-1) and 0.002-0.0075 ng mL(-1), respectively. A 20-fold dilution of milk samples prevented a matrix effect of the milk and allowed measurement of AFM1 at concentrations below than the maximum acceptable limit. Values of recovery within and between assays were 81.5-117.6% and 86-110.6%, respectively. The results of using the developed CL-ELISA to analyse samples of six brands of milk that were purchased in Taiwan revealed that AFM1 was absent from all studied samples. PMID:24731347

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

2014-09-01

137

Determination of residual ractopamine concentrations by enzyme immunoassay in treated pig's tissues on days after withdrawal.  

PubMed

The objective of this study was to measure residual ractopamine concentrations in tissues of pigs as experimental animals after treatment with dietary ractopamine for 28 consecutive days. Ractopamine was administered orally to the experimental group (n=9) in a dose of 0.1mg/kg body mass per day, whereas control animals (n=3) were left untreated. Treated pigs (60kg) were sacrificed on days 1, 3 and 8 of treatment discontinuation and residues were determined in kidney, liver, muscle, brain and heart tissues using previously validated enzyme-linked immunosorbent assay (ELISA) as a quantitative screening method. Validation showed good mean recoveries (approx. 70-90%) with acceptable inter- and intra-day relative standard deviations (RSD<13%), demonstrating the method efficiency in determination of ractopamine tissue concentrations. The highest ractopamine concentration on day 1 (24h) after the last exposure was recorded in the kidney (12.49±7.96ng/g), followed by the liver (7.21±2.73ng/g), heart (1.26±0.12ng/g) and brain (0.63±0.05ng/g); at this time of withdrawal, residues were not detected in the muscle. Ractopamine was depleted rapidly from all study tissues, with mostly no detectable residues on day 8 of withdrawal. PMID:22119670

Pleadin, Jelka; Perši, Nina; Vuli?, Ana; Mili?, Dinka; Vah?i?, Nada

2012-03-01

138

Development and Application of an Immunoaffinity Column Enzyme Immunoassay for Mycotoxin Zearalenone in Complicated Samples  

PubMed Central

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC50 against ZEA of 0.02 µg L?1. The mAb 2D3 exhibited a high recognition of ZEA (100%) and ?-zearalenol (?-ZOL, 88.2%). Its cross-reactivity with ?-zearalenol (?-ZOL) and ?-zearalanol (?-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize. PMID:24465616

Li, Peiwu; Zhang, Qi; Li, Ran; Zhang, Wen; Ding, Xiaoxia; Lei, Jiawen; Zhang, Zhaowei

2014-01-01

139

Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea.  

PubMed Central

Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use. The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C. difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C. difficile Toxin A; bioMérieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C. difficile-associated diarrhea (CDAD). The study was performed with 285 fresh stools from 285 patients with suspected CDAD. In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed. CDAD diagnosis was established in 55 cases (incidence, 19.3%). The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas. The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas. We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result. Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test. PMID:8463404

Barbut, F; Kajzer, C; Planas, N; Petit, J C

1993-01-01

140

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

PubMed Central

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

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

2014-01-01

141

The superiority of polymerase chain reaction over an amplified enzyme immunoassay for the detection of genital chlamydial infections  

PubMed Central

Background/objectives The polymer conjugate enhanced enzyme immunoassay (IDEIA) and Cobas Amplicor polymerase chain reaction Chlamydia trachomatis (CT) (Amplicor PCR) are two commonly used assays for the diagnosis of CT infection. The performance of these assays was compared for the diagnosis of genital CT infection among 1000 consecutive patients attending a genitourinary medicine (GUM) clinic. Confirmation of positive results and the clinical significance of the absence of cryptic plasmid in chlamydia on the diagnosis of infection by Amplicor PCR were also investigated. Methods IDEIA, Amplicor PCR, and two nested in?house PCR assays targeting cryptic plasmid and omp1 gene were performed on all samples. DNA from Amplicor PCR negative samples was pooled for in?house PCR assays. Each pool contained DNA from seven Amplicor PCR negative samples. Results Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of IDEIA in the diagnosis of genital CT infection were 80%, 97%, 80%, 97%, and 95%, respectively. Sensitivity, specificity, PPV, NPV and efficiency of Amplicor PCR were 99%, 98%, 89%, 100%, and 98%, respectively. 16 (11%) of 144 Amplicor PCR positive results were identified as false positive by in?house PCR assays. No isolate of plasmid free CT was detected among the study population. Conclusions IDEIA should not be used for the diagnosis of CT infection because of its poor sensitivity. Although the analytic specificity of Amplicor PCR was 98%, because of the adverse medical, social, and psychological impact of false positive results for patients, confirmation of Amplicor PCR positive results by a different assay with comparable sensitivity is essential. Amplification assays targeting cryptic plasmid are appropriate for the diagnosis of genital CT infections. PMID:16461600

Jalal, H; Stephen, H; Al-Suwaine, A; Sonnex, C; Carne, C

2006-01-01

142

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection  

PubMed Central

Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care. PMID:22189114

Sural, Preethi; Loomis, Caroline R.; Pesta, Christine; Gonzalez-Krellwitz, Laura; Robinson-Dunn, Barbara; Riska, Paul

2012-01-01

143

Evaluation of three multiplex flow immunoassays compared to an enzyme immunoassay for the detection and differentiation of IgG class antibodies to herpes simplex virus types 1 and 2.  

PubMed

The diagnosis of herpes simplex virus (HSV) infections is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture. However, in instances of subclinical or unrecognized HSV infection, serologic testing for IgG class antibodies to type-specific HSV glycoprotein G (gG) may be useful. This study evaluated and compared the performances of three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) for the simultaneous detection of gG type-specific IgG antibodies to HSV types 1 and 2 (HSV-1 and HSV-2). Serum specimens (n = 505) submitted for routine gG type-specific HSV IgG testing by enzyme immunoassay (EIA) (HerpeSelect; Focus Diagnostics) were also tested by the three multiplex flow immunoassays. Specimens showing discordant results were tested by HSV type-specific Western blotting (WB). For HSV-1 IgG, the AtheNA, BioPlex, and Plexus assays demonstrated agreements of 94.9% (479/505 specimens), 97.8% (494/505 specimens), and 97.4% (492/505 specimens), respectively, with the results of EIA. For HSV-2 IgG, the AtheNA, BioPlex, and Plexus assays showed agreements of 87.9% (444/505 specimens), 97.2% (491/505 specimens), and 96.8% (489/505 specimens), respectively, with EIA results. Timing studies showed that the AtheNA, BioPlex, and Plexus assays could provide complete analysis of 90 serum specimens in 3.1, 1.5, and 2.9 h, respectively, versus 3.1 h by EIA. These findings suggest that the gG type-specific HSV IgG multiplex immunoassays may be beneficial to high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for HSV serologic testing. The evaluated systems provide comparable results to those of EIA, while reducing hands-on time and eliminating the necessity to aliquot specimens prior to testing. PMID:20007366

Binnicker, M J; Jespersen, D J; Harring, J A

2010-02-01

144

Immunoassay Animations  

NSDL National Science Digital Library

This site features animations showing the detailed steps involved in eight different immunoassay examples. The focus of the site is primarily on the biochemical aspects of the immunoassays, not on their analytical applications. The animations depict the following immunoassays: Dihydroxy Vitamin D, ACTH, BoneĂ­specific Alkaline Phosphatase, Cortisol, Deoxypyridinoline, Osteocalcin, Prolactin and Thyroxine.

Chung, Kyn W.

2011-05-24

145

Comparison among the BED capture enzyme immunoassay test and AxSYM avidity index assay for determining recent HIV infection and incidence in two Voluntary Counselling and Testing Centres in Northeast Brazil.  

PubMed

The aims of this study were to compare the automated AxSYM avidity assay index with the BED capture enzyme immunoassay test and to calculate the HIV-1 incidence using the BED capture enzyme immunoassay and AxSYM avidity assay index algorithms within a population seeking the Voluntary Counselling and Testing Centres in two municipalities in the Metropolitan Region of Recife, Northeast of Brazil. An analysis was conducted in 365 samples that tested positive for HIV infection from frozen serum collected during the period 2006-2009. There was a similar proportion of males and females; most patients were heterosexual (86%) with a median age of 29 years. Of the 365 samples, 102 (28%) and 66 (18.1%) were identified as recent infections by BED capture enzyme immunoassay and AxSYM avidity assay index, respectively. The HIV-1 total incidence in the BED capture enzyme immunoassay and AxSYM avidity assay index algorithms were: 0.79 (95% CI: 0.60-0.98) and 0.34 (95% CI: -0.04 to 0.72), respectively. Incidence was higher among men. There was good agreement between the tests, with a kappa of 0.654 and a specificity of 95.8%. AxSYM avidity assay index may be helpful in improving the quality of the estimates of recent HIV infection and incidence, particularly when used in a combined algorithm with BED capture enzyme immunoassay. PMID:24780363

Salustiano, Daniela Medeiros; Lima, Kledoaldo Oliveira de; Cavalcanti, Ana Maria Salustiano; Diaz, Ricardo Sobhie; Lacerda, Heloisa Ramos

2014-01-01

146

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection  

Microsoft Academic Search

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and

Abdelfattah M. Attallah; Hisham Ismail; Gellan G. Ibrahim; Mohamed Abdel-Raouf; Ahmed M. El-Waseef; Mohamed Abdel-Wahab

2004-01-01

147

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.  

PubMed

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination. PMID:1750684

MacLean, A I; Bachas, L G

1991-06-01

148

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

PubMed

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

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

2009-01-01

149

Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.  

PubMed

This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (? = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody. PMID:24785462

Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

2014-05-20

150

Toxicity comparison of chlorinated and brominated dibenzo-p-dioxins and dibenzofurans in industrial source samples by HRGC/HRMS and enzyme immunoassay.  

PubMed

Limited information is available on the applicability of polychlorinated dibenzo-p-dioxin/furan (PCDD/F) toxicity assays to their brominated counterparts: polybrominated dibenzo-p-dioxins/furans (PBDDs/Fs). We estimated the toxicity of mixtures of chlorinated, brominated, and mixed bromochloro-dioxins and -furan (PBCDDs/Fs) laboratory standards using a chemically-activated luciferase gene expression cell bioassay (CALUX). The relative effects potency (REP) values obtained were comparable to the World Health Organization (WHO) toxic equivalency factors (TEFs) and in agreement with the concept of additive congener toxicity of mixtures of dioxins and furans. Enzyme immunoassay (EIA)-based toxic equivalents (TEQs), however, showed overestimation for PCDDs/Fs (0-4 orders of magnitudes higher) and underestimation for PBDDs/Fs (0-1 orders of magnitude lower) when compared to high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS)-based TEQ calculation (using WHO TEFs) in samples from an industrial source line. No correlation was found between the EIA and the HRGC/HRMS data, which could be attributed to differences in homologue-specific cross-reactivity responses, sample matrix type, and presence of other compounds competing for antibody binding in the immunoassay. PMID:20110126

Samara, Fatin; Wyrzykowska, Barbara; Tabor, Dennis; Touati, Dahman; Gullett, Brian K

2010-04-01

151

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

SciTech Connect

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

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

2006-06-16

152

Immunoassay Animations  

NSDL National Science Digital Library

The University of Glasgow Department of Pathological Biochemistry has recently made available five immunoassay animations that draw on the interactivity of the FutureSplash plug-in (discussed in the December 20, 1996 issue of the Scout Report). The animations are "a learning resource for students, to show the wide application of the use of antibodies in a clinical biochemistry laboratory," and are "graphical representations of the immunoassay methodology used by a number of commercial manufacturers." Each immunoassay is presented as a series of animations, allowing the user to navigate forward and back in time. A key is provided, and animations can be viewed step by step (with explanations) and then replayed as a single continuous animation without explanations or navigation. Immunoassay Animations is a powerful visual teaching tool.

Chung, Kynwai.; Cowan, Bob.

1996-01-01

153

Supernovae and the IGM  

E-print Network

An energetic argument implies that a galaxy like the Milky Way is blowing a powerful wind that carries away most of the heavy elements currently synthesized and has impacted the IGM out to at least 180 kpc. Rich clusters of galaxies appear to be closed systems in which most heavy elements are ejected from galaxies. More supernovae are required than the yield of core-collapse SNe from a Salpeter IMF. X-ray observations imply that the IGM in groups and clusters as been strongly preheated. SNe probably cannot supply the required energy, which must come from AGN.

James Binney

2000-08-23

154

Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment.  

PubMed

The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml(-1), recoveries were 80-128% at 4-40 ng ml(-1). Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ? 20 ng ml(-1)) and a receptor assay (ßeta-s.t.a.r., detection limit ? 15 ng ml(-1)) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10-27 ?g?ml(-1)) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml(-1)). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 ?g kg(-1)) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material. PMID:21103866

Thal, Johannes; Steffen, Monika; Meier, Bianca; Schneider, Elisabeth; Adriany, Ansgar; Usleber, Ewald

2011-01-01

155

The Occurrence of Abscisic Acid and Abscisyl-?-d-Glucopyranoside in Developing and Mature Citrus Fruit as Determined by Enzyme Immunoassay 1  

PubMed Central

The contents of (+)-cis-abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate(s) were analyzed by means of enzyme immunoassay in partially purified extracts of developing and mature sweet orange fruit (Citrus sinensis [L.] Osbeck cv Washington navel). A relatively small increase in ABA was observed in the fruit exocarp during the natural color transition from green to orange. At the same time, the ABA-conjugate level increased approximately 12-fold in this tissue. The contents of ABA and ABA-conjugate equaled 15.0 ± 0.7 and 107.8 ± 2.1 nanomoles per gram fresh weight, respectively, in the exocarp at harvest. Other tissues also contained considerable quantities of these compounds. Whereas the highest ABA content was observed in the exocarp, the highest ABA-conjugate content was observed in the central vascular axis of the fruit and equaled 187.0 ± 10.3 nanomoles per gram fresh weight. The only immunoreactive conjugate found in significant quantity in mature fruit was identified as abscisyl-?-d-glucopyranoside (ABA-GE) based on (a) immunological cross-reactivity, (b) thin layer chromatography co-chromatography with authentic standards in two solvent systems, (c) susceptibility to both chemical and enzymic degradation, and (d) mass spectroscopy. PMID:16665032

Harris, Michael J.; Dugger, William M.

1986-01-01

156

EQCM immunoassay for phosphorylated acetylcholinesterase as a biomarker for organophosphate exposures based on selective zirconia adsorption and enzyme-catalytic precipitation.  

PubMed

A zirconia (ZrO(2)) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (Phospho-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 ZrO(2) film with uniform nanostructures. The resulting ZrO(2) film was utilized to selectively capture Phospho-AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated proteins. 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 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 Phospho-AChE in human plasma with a detection limit of 0.020 nM. 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. PMID:19135350

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

2009-04-15

157

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

PubMed

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 ?M), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination. PMID:25079057

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

2014-09-01

158

Ultrasensitive immunoassay techniques.  

PubMed

Ultrasensitive detection of clinically important substances using assays based on ligand:binder interactions has revolutionized laboratory medicine. Various strategies have been perfected to push the analytical sensitivity of ligand:binder assays (e.g., immunoassay, blotting, nucleic acid hybridization assay) into the attomole and zeptomole region (10(-18)-10(-21) mol). These include the use of labels with amplifying properties (e.g., enzymes), multiple labeling, and cascade reactions. In addition a wide range of ultrasensitive luminescent detection reactions have been developed for conventional enzyme labels based on chemiluminescent, bioluminescent, and time-resolved fluorescent reactions. PMID:8299202

Kricka, L J

1993-10-01

159

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

PubMed Central

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

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

1984-01-01

160

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children  

PubMed Central

Sapoviruses (SVs) are an important cause of acute pediatric gastroenteritis. Due to the lack of appropriate diagnostic methods, the epidemiology of SV-associated illness remains poorly understood. Baculovirus and Escherichia coli expression systems were evaluated for the development of antibody detection enzyme immunoassays (EIA). Age-related antibody prevalence in children was studied using the new EIA. Because of the low yield of the baculovirus system, the E. coli-expressed SV capsid proteins were used to develop the EIA. The antigenic specificities of the E. coli-expressed SV capsid proteins were demonstrated using hyperimmune antisera raised in animals and sera collected from patients. A high prevalence (>90%) of antibodies to both SV (strain Mex340) and norovirus (strain VA387) was observed in children involved in a birth cohort at 20 to 24 months of age; however, at 1 to 3 months of age, <25% of the children possessed anti-SV antibodies versus >90% with anti-NV antibodies. The E. coli-derived SV proteins are an excellent source of antigens for the EIA. SV infection is common in the first 2 years of life. The low prevalence of maternal antibodies detected in Mexican children against SVs in this study is unique and needs to be addressed in future studies. PMID:17021096

Farkas, Tibor; Deng, Xiaoyun; Ruiz-Palacios, Guillermo; Morrow, Ardythe; Jiang, Xi

2006-01-01

161

[Development of an enzyme immunoassay for the detection of antibodies from hens and mice against Newcastle disease virus].  

PubMed

The development of an enzyme-immuno-assay (EIA) for detection of avian and mouse antibodies against the Newcastle-Disease Virus is shown. The antigen preparation was the centre of the experiments. The basic assay programme and selected test steps were optimized. The established assay is part of the EIA-control system for a SPF (specific-pathogen-free)-flock and also the essential screening method for the production of anti-NDV-monoclonal antibodies. PMID:8512547

Minning, P; Hlinak, A; Klaczinski, K

1993-05-01

162

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

Microsoft Academic Search

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

Cormac Kilty; Sean Doyle; Brian Hassett; Fiona Manning

1998-01-01

163

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

PubMed

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 ?g mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 ?g mL(-1)) and by the Directive 2004/19/EC (0.6 ?g mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays. PMID:24223419

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

2014-01-01

164

Use of a monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens.  

PubMed

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool. PMID:2860814

Ungar, B L; Yolken, R H; Quinn, T C

1985-05-01

165

Survey of immunoassay techniques for biological analysis  

SciTech Connect

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.

Burtis, C.A.

1986-10-01

166

An efficient sample preparation method for high-throughput analysis of 15(S)-8-iso-PGF2? in plasma and urine by enzyme immunoassay.  

PubMed

Although several methods have been reported on the analysis of the oxidative stress marker 15(S)-8-iso-prostaglandin-F2alpha (8-iso-PGF2?) in biological fluids, they either involve extensive sample preparation and costly technology or require high sample volume. This study presents a sample preparation method that utilizes low sample volume for 8-iso-PGF2? analysis in plasma and urine by an enzyme immunoassay (EIA). In brief, 8-iso-PGF2? in deproteinized plasma or native urine sample is complexed with an antibody and then captured by molecular weight cut-off filtration. This method was compared with two other sample preparation methods that are typically used in the analysis of 8-iso-PGF2? by EIA: Cayman's affinity column purification method and solid-phase extraction on C-18. The immunoaffinity purification method described here was superior to the other two sample preparation methods and yielded recovery values of 99.8 and 54.1% for 8-iso-PGF2? in plasma and urine, respectively. Analytical precision (relative standard deviation) was ±5% for plasma and ±15% for urine. The analysis of healthy human plasma and urine resulted in basal 8-iso-PGF2? levels of 31.8 ± 5.5 pg/mL and 2.9 ± 2.0 ng/mg creatinine, respectively. The robustness and analytical performance of this method makes it a promising tool for high-throughput screening of biological samples for 8-iso-PGF2?. PMID:22989424

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

2012-01-01

167

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

PubMed

The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days. PMID:24257195

Kajaysri, Jatuporn; Nokkaew, Weerapun

2014-03-01

168

A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.  

PubMed

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [kappa = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars. PMID:17872971

Quint, Koen D; van Doorn, Leen-Jan; Kleter, Bernhard; de Koning, Maurits N C; van den Munckhof, Henk A M; Morre, Servaas A; ter Harmsel, Bram; Weiderpass, Elisabete; Harbers, Gonneke; Melchers, Willem J G; Quint, Wim G V

2007-11-01

169

Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus.  

PubMed Central

Influenza A virus infections are a major cause of morbidity and mortality worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivity. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcription PCR was performed with oligonucleotide primers directed at a highly conserved area of the influenza A matrix gene. Amplified DNA was identified by hybridization in solution to a nested biotinylated RNA probe and quantitated in an EIA. PCR-EIA detected small quantities of RNA from the three prevalent subtypes of human influenza A virus. Influenza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use in clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. Thirty-seven of the postinfection samples had detectable influenza A virus RNA by PCR-EIA, whereas only 26 postinfection samples were positive by culture. PCR-EIA was particularly efficient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were positive, whereas virus was cultivated from 4 samples (P < 0.00005). All preinfection samples from volunteers subsequently infected with influenza A virus were negative by PCR-EIA, as were samples from a volunteer infected with parainfluenza virus type 3. Nucleic acid amplification techniques represent important tools for the timely and sensitive diagnosis of influenza A virus infections and, therefore, their management and control. Images PMID:8195369

Cherian, T; Bobo, L; Steinhoff, M C; Karron, R A; Yolken, R H

1994-01-01

170

Assessment of Pregnancy Status of Asian Elephants (Elephas maximus) by Measurement of Progestagen and Glucocorticoid and Their Metabolite Concentrations in Serum and Feces, Using Enzyme Immunoassay (EIA)  

PubMed Central

ABSTRACT The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days. PMID:24257195

KAJAYSRI, Jatuporn; NOKKAEW, Weerapun

2013-01-01

171

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).  

PubMed

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

Pribbenow, Susanne; Jewgenow, Katarina; Vargas, Astrid; Serra, Rodrigo; Naidenko, Sergey; Dehnhard, Martin

2014-09-15

172

New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA  

PubMed Central

A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 ?g/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed. PMID:12734224

Kimura, Tatsuji; Rokuhara, Akinori; Matsumoto, Akihiro; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Maki, Noboru

2003-01-01

173

Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus.  

PubMed

Influenza A virus infections are a major cause of morbidity and mortality worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivity. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcription PCR was performed with oligonucleotide primers directed at a highly conserved area of the influenza A matrix gene. Amplified DNA was identified by hybridization in solution to a nested biotinylated RNA probe and quantitated in an EIA. PCR-EIA detected small quantities of RNA from the three prevalent subtypes of human influenza A virus. Influenza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use in clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. Thirty-seven of the postinfection samples had detectable influenza A virus RNA by PCR-EIA, whereas only 26 postinfection samples were positive by culture. PCR-EIA was particularly efficient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were positive, whereas virus was cultivated from 4 samples (P < 0.00005). All preinfection samples from volunteers subsequently infected with influenza A virus were negative by PCR-EIA, as were samples from a volunteer infected with parainfluenza virus type 3. Nucleic acid amplification techniques represent important tools for the timely and sensitive diagnosis of influenza A virus infections and, therefore, their management and control. PMID:8195369

Cherian, T; Bobo, L; Steinhoff, M C; Karron, R A; Yolken, R H

1994-03-01

174

Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant H(C) subunit of botulinum neurotoxin type A monoclonal antibodies.  

PubMed

Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H(C) subunit of botulinum neurotoxin type A (rAH(C)) was expressed as an effective vaccine against botulism, indicating that the rAH(C) could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH(C), 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH(C) and BoNT/A reached 0.45 pg mL(-1). This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20-40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A. PMID:22713913

Liu, Zhijia; Song, Chaojun; Li, Yongming; Liu, Fei; Zhang, Kui; Sun, Yuanjie; Li, Haitao; Wei, Yuying; Xu, Zhuwei; Zhang, Chunmei; Yang, Angang; Xu, Zhikai; Yang, Kun; Jin, Boquan

2012-07-20

175

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

PubMed

A competitive enzyme-linked immunosorbent assay (ELISA) for fenoxycarb was adapted for quantitative detection of this insect growth regulator in various environmental, agricultural, food and biological matrices. Environmental samples were taken from soil and surface waters in Hungary. The ELISA enabled fenoxycarb detection in surface waters in the 1.1-125 ng ml(-1) concentration range without sample cleanup. In contrast, soil produced a strong matrix effect due to humic acids and other soil components. Several fruit homogenates and commercial fruit juices (eg apple, pear, grape) were analyzed by the ELISA. The assay was found to be suitable for analysis of fenoxycarb in fruit juices diluted 1:40. Biological samples included insect, fish and bovine tissues. The ELISA was applied to detect fenoxycarb in various biological matrices from larvae of the silkworm, Bombyx mori L. The assay proved useful for the analysis of haemolymph diluted 1:10 or at higher dilutions. Fat body and whole body homogenates, however, caused severe matrix effects. Fenoxycarb was detected in liver homogenates (diluted 1:40) from fish treated with various doses of fenoxycarb, and the concentrations determined correlated with the applied doses. The method was used to analyze spiked bovine urine samples diluted 1:10 or at greater dilutions. Fenoxycarb content determined by the ELISA in water and fruit juice samples was validated using GC-MS with solid-phase microextraction (SPME) sample preparation. The results of these studies demonstrated both the value and limitations of the assay when used for monitoring fenoxycarb in environmental, food and biological samples. PMID:12701701

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

2003-04-01

176

An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products  

PubMed Central

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL?1) ELISA format, in which the antibody was diluted to 1?80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g?1 for wheat, 0.06 ng·g?1 for maize, and 0.1 ng·g?1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

2014-01-01

177

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.  

PubMed Central

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results. Images PMID:1757540

DiPersio, J R; Varga, F J; Conwell, D L; Kraft, J A; Kozak, K J; Willis, D H

1991-01-01

178

Bioelectrochemical Immunoassay of Polychlorinated Biphenyl  

SciTech Connect

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.

Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

2008-04-01

179

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

E-print Network

APPENDIX B ILLUSTRATION OF IMMUNOASSAY TECHNIQUES. . APPENDIX C ILLUSTRATION OF A 2-STEP ELISA. . . . . . . . . . 54 58 APPENDIX D ILLUSTRATION OF HP5995C/96A GC/MS SYSTEM. . 60 APPENDIX E CALIBRATION SETUP FOR SAMPLING PUMP. . APPENDIX F OSHA... METHODOLOGY FOR PYRETHRUM. . . . ~ ~ ~ APPENDIX G COMPOSITE LISTING OF MATERIALS. ~ ~ . . . ~ 62 65 91 APPENDIX H 2-STEP COMPETITION ELISA FOR PERMETHRIN. . . 93 APPENDIX I GAS CHROMATOGRAM/MASS SPECTRUMS' 97 APPENDIX J FIGURES. APPENDIX K TABLES...

Ball, Madalyn Eunice

2012-06-07

180

Multiplexed single molecule immunoassays.  

PubMed

We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells--or Single Molecule Arrays (Simoa)--that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-?, IL-6, IL-1?, and IL-1? in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease. PMID:23719780

Rissin, David M; Kan, Cheuk W; Song, Linan; Rivnak, Andrew J; Fishburn, Matthew W; Shao, Qichao; Piech, Tomasz; Ferrell, Evan P; Meyer, Raymond E; Campbell, Todd G; Fournier, David R; Duffy, David C

2013-08-01

181

Detection of anticardiolipin antibody igm by sm(3+)-labeled time-resolved fluoroimmunoassay.  

PubMed

In an effort to improve the quantitative detection of aCL IgM, we develop a new immunoassay to improve aCL IgM detection based on TRFIA using the complex of cardiolipin plus bovine ?2GPI as antigen and Sm(3+)-labeled rabbit anti-human IgM as conjugate. The precision, sensitivity, specificity, coefficient of recovery, and stability of the assay were evaluated and comparison with the classical ELISA was also made. The aCL IgM TRFIA kit we established had a wider detectable range than commercial ELISA ones when diluted a specimen with strong positive from 1:2.5-1:40960. We observed that for the established TRFIA kit there was a good liner range within 1:2.5-1:40960, whereas it was within 1:20-1:1280 when using ELISA kits. The intraassay precision rate and the interassay precision rate were <5% for 3 different concentrations. The sensitivity was 0.1MPL U/mL and the clinical diagnostic specificity was 98%. Average recovery rate was 101.13%. The established assay kit also behaved better in stability. Additionally, the immunoassay we established correlated well with the ELISA and the correlation coefficient was 0.956. We thus conclude that the TRFIA we developed for aCL IgM detection gives promise to a more sensitivity and reliable diagnosis of APS and has potential value for large-scale screening programs. PMID:23656246

Hu, Zhigang; Liu, Jie; Ye, Yan; Zhou, Yaohong; Yu, Lei

2013-01-01

182

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)

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).

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

2008-10-01

183

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 ...

184

Immunoassay as a screening tool for industrial toxicants  

SciTech Connect

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.

Pierce, T.

1986-08-01

185

Food allergen detection with biosensor immunoassays.  

PubMed

An optical biosensor was used to develop both direct and sandwich immunoassays for the detection of proteins from milk, egg, hazelnut, peanut, shellfish, and sesame in food samples. Affinity-purified polyclonal antibodies raised against the proteins were immobilized on the biosensor chip. Food samples were injected and the proteins that bound to the antibodies on the surface were detected by a shift in the resonance angle. By adding a second antibody in a sandwich assay, matrix effects could be overcome and the sensitivity and selectivity enhanced. Detection of allergen levels down to 1-12.5 microg/g in food samples was demonstrated for the various assays. Good agreement of results was also obtained from parallel analysis with alternative immunoassays, including rocket immunoelectrophoresis, enzyme immunoassay, and immunoblotting. The present study demonstrates that the sensitivity of the described biosensor technique is comparable to the most sensitive enzymed-linked immunosorbent assays. PMID:16792086

Yman, Ingrid Malmheden; Eriksson, Anders; Johansson, M Annette; Hellenäs, Karl-Erik

2006-01-01

186

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

PubMed Central

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

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

2013-01-01

187

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...

188

Rubella-Specific IgM Antibodies in Postnatal Infection  

Microsoft Academic Search

A total of 214 serum specimens of postnatal rubella infected cases were determined for persistence of rubella-specific IgM antibodies, using solid-phase immunosorbent hemadsorp­ tion (SPIHAd) test and enzyme-linked immunosorbent assay (ELISA). Initially, 148 blood samples were obtained from 77 patients comprising 74 females and 3 males. All patients were clinical rubella cases whose sera were confirmed by serodiagnosis. The remaining

Uraiwan Kositanont; Pilaipan Puthavathana; Chantapong Wasi

189

An immunoassay for atrazine using tunable immunosorbent Jae-Young Kim,a,b  

E-print Network

synthesized biologically and purified by temperature-triggered phase transition. A competitive immunoassay in immunoassay [12,13]. Enzyme-linked immuno- sorbent assay (ELISA)1 has been widely used in envi- ronmental Abbreviations used: ELISA, enzyme-linked immunosorbent assay; ELP, elastin-like polypeptide; 2,4-D, 2

Chen, Wilfred

190

Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene.  

PubMed

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies. PMID:9705390

Becker, K; Roth, R; Peters, G

1998-09-01

191

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 Central

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

McGowan, Karin L.

2012-01-01

192

Microfluidic chips for immunoassays.  

PubMed

The use of microfluidic chips for immunoassays has been extensively explored in recent years. The combination of immunoassays and microfluidics affords a promising platform for multiple, sensitive, and automatic point-of-care (POC) diagnostics. In this review, we focus on the description of recent achievements in microfluidic chips for immunoassays categorized by their detection method. Following a brief introduction to the basic principles of each detection method, we examine current microfluidic immunosensor detection systems in detail. We also highlight interesting strategies for sensitive immunosensing configurations, multiplexed analysis, and POC diagnostics in microfluidic immunosensors. PMID:23495732

Han, Kwi Nam; Li, Cheng Ai; Seong, Gi Hun

2013-01-01

193

Serum Free Light Chain Only Myeloma with Cytoplasmic IgM  

PubMed Central

In the past decade, the serum free light chain (FLC) immunoassays have become widely available enabling greater sensitivity in the diagnosis and management of monoclonal light chain diseases. Here, we describe a rare case of serum free light chain only myeloma with cytoplasmic IgM. A 75-year-old woman presented with a progressively worsening lumbosacral pain. FDG PET/CT images showed increased FDG uptake in the sacral mass, vertebral bodies, and ribs. Laboratory data found hypogammaglobulinemia and the bone marrow aspirate revealed only 2.2% of plasma cells. The serum and urine protein electrophoresis did not detect a monoclonal band. However, the serum FLC immunoassays reported an abnormal kappa/lambda ratio (0.001) indicating the presence of monoclonal lambda FLC. The sacral tumor biopsy revealed proliferation of plasma cells and immunohistochemical staining showed that the plasma cells were positive for CD138, IgM, and lambda light chain but negative for CD20. This case may have previously been described as a nonsecretory IgM myeloma but recently would be identified as free light chain only myeloma. The immunohistochemical and genetic features of the clonal plasma cells in free light chain only myeloma need to be further investigated to better understand the relevance and incidence of this myeloma type. PMID:25028614

Ebana, Hideaki; Nakamura, Ken-ichi; Nozawa, Yoshihiro; Seki, Ritsuko

2014-01-01

194

Serum Free Light Chain Only Myeloma with Cytoplasmic IgM.  

PubMed

In the past decade, the serum free light chain (FLC) immunoassays have become widely available enabling greater sensitivity in the diagnosis and management of monoclonal light chain diseases. Here, we describe a rare case of serum free light chain only myeloma with cytoplasmic IgM. A 75-year-old woman presented with a progressively worsening lumbosacral pain. FDG PET/CT images showed increased FDG uptake in the sacral mass, vertebral bodies, and ribs. Laboratory data found hypogammaglobulinemia and the bone marrow aspirate revealed only 2.2% of plasma cells. The serum and urine protein electrophoresis did not detect a monoclonal band. However, the serum FLC immunoassays reported an abnormal kappa/lambda ratio (0.001) indicating the presence of monoclonal lambda FLC. The sacral tumor biopsy revealed proliferation of plasma cells and immunohistochemical staining showed that the plasma cells were positive for CD138, IgM, and lambda light chain but negative for CD20. This case may have previously been described as a nonsecretory IgM myeloma but recently would be identified as free light chain only myeloma. The immunohistochemical and genetic features of the clonal plasma cells in free light chain only myeloma need to be further investigated to better understand the relevance and incidence of this myeloma type. PMID:25028614

Ebana, Hideaki; Nakamura, Ken-Ichi; Nozawa, Yoshihiro; Seki, Ritsuko; Mita, Masayuki

2014-01-01

195

Automated HomogeneousLiposome ImmunoassaySystems for Anticonvulsant Drugs  

Microsoft Academic Search

0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx#{174}) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsantdrug con- centrations in blood. AdditionalKeyphrases:enzyme immunoassay fluorescence polarizationimmunoassay Phenytoin (PHT), phenobarbital (PB), and carba- mazepine (CBZ) are widely used in the treatment of epileptic disorders.4 These drugs have narrow therapeu- tic ranges,

Kazuhisa Kubotsu; Sachiko Goto; Mmoru Fujita; Hideaki Tuchiya; Masaaki Kida; Susumu Takano; Shuji Matsuura; Ikunosuke Sakurabayashi

196

Lanthanide-based time-resolved luminescence immunoassays  

Microsoft Academic Search

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications,\\u000a for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by\\u000a use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem\\u000a of the background

A. K. Hagan; T. Zuchner

2011-01-01

197

Reliable confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) with an enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, pol, and env genes.  

PubMed Central

An enzyme-linked immunoassay (ELISA) using six recombinant proteins corresponding to large segments of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env gene products (HIVAGEN; SmithKline Bio-Science Laboratories, Van Nuys, Calif.) was developed to confirm the presence of antibodies to HIV-1 in sera reactive in the whole-cell-derived virion screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from the different categories of HIV-infected individuals (asymptomatic, acquired immunodeficiency syndrome [AIDS]-related complex, and AIDS). A positive reaction was defined as reactivity against an env and at least one other (either gag or pol) HIV-1 gene product; negative was defined as no reaction with any antigen; and indeterminate was defined as reactivity with gag or pol (or both) or with env alone. None of the 1,180 serum samples from healthy seronegative blood donors gave a positive result, and only 49 of these samples (4%) gave indeterminate results. The recombinant HIV-1 antigen ELISA panel identified seropositive individuals with a high degree of accuracy, as a positive reaction was seen with 99.3% of asymptomatic healthy seropositive individuals, 98.1% of patients with AIDS-related complex, and 90.4% of patients with AIDS. None of the 725 HIV-1-seropositive subjects had a negative test result. Reactivity with the Kp41 antigen, corresponding to an amino-terminal portion of the gp41 envelope glycoprotein, by itself demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. A subset of seronegative and seropositive samples were tested both with the recombinant HIV-1 antigen ELISA panel and by Western blot (Du Pont Co.). The recombinant HIV-1 antigen ELISA panel accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did Western blot (interpreted by Du Pont criteria). PMID:2787334

Ng, V L; Chiang, C S; Debouck, C; McGrath, M S; Grove, T H; Mills, J

1989-01-01

198

Carbon nanotube-based transducers for immunoassays  

Microsoft Academic Search

The attachment of mouse immunoglobulin G (IgG) and anti-mouse IgG antibodies onto carbon nanotubes (CNTs), using either non-covalent or covalent means was investigated. The resultant CNTs were characterised using a variety of techniques including enzyme-linked and fluorescence-linked immunoassays, UV–visible-NIR and Raman spectroscopy, transmission electron microscopy and cyclic voltammetry. TEM images of the adsorbed antibody on the CNTs show that the

Carol Lynam; Niamh Gilmartin; Andrew I. Minetta; Richard O’Kennedy; Gordon Wallace

2009-01-01

199

Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil  

PubMed Central

Background American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. Methods A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. Results The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. Interpretation ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil. PMID:23922884

de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

2013-01-01

200

Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.  

PubMed

A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin-ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC50) at 0.12 ?g kg(-1) for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 ?g kg(-1) and the LODs for CIP and ENR were all <0.2 ?g kg(-1). Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 ?g kg(-1) for PEF to 2.1 ?g kg(-1) for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 ?g kg(-1)) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool. PMID:23842902

Tao, Xiaoqi; Chen, Min; Jiang, Haiyang; Shen, Jianzhong; Wang, Zhanhui; Wang, Xia; Wu, Xiaoping; Wen, Kai

2013-09-01

201

Interferences in Immunoassay  

PubMed Central

Substances that alter the measurable concentration of the analyte or alter antibody binding can potentially result in immunoassay interference. Interfering, endogenous substances that are natural, polyreactive antibodies or autoantibodies (heterophiles), or human anti-animal antibodies together with other unsuspected binding proteins that are unique to the individual, can interfere with the reaction between analyte and reagent antibodies in immunoassay. Lipaemia, cross-reactivity, and exogenous interferences due to pre-analytical variation, matrix and equipment reaction also affect immunoassay. Interfering substances may lead to falsely elevated or falsely low analyte concentration in one or more assay systems depending on the site of the interference in the reaction and possibly result in discordant results for other analytes. The prevalence of interference is generally low in assays containing blocking agents that neutralise or inhibit the interference but is often higher in new, untested immunoassays. A wide range of analytes measured by immunoassay including hormones, tumour markers, drugs, cardiac troponin and microbial serology may be affected. Interference in immunoassay may lead to the misinterpretation of a patient's results by the laboratory and the wrong course of treatment being given by the physician. Laboratories should put processes in place to detect, test and report suspected interferences. It is equally important that physicians communicate any clinical suspicion of discordance between the clinical and the laboratory data to the laboratory. The detection of interference may require the use of an alternate assay or additional measurements, before and after treatment with additional blocking reagent, or following dilution of the sample in non-immune serum. It is imperative that laboratories inform physicians of the follow-up procedure and report on the presence of any interference. The establishment of on-going laboratory-physician contact is essential to the continuing awareness of wrong patient results due to interference. PMID:18458713

Tate, Jill; Ward, Greg

2004-01-01

202

New trends in immunoassays.  

PubMed

This article takes a special focus on signal amplification technologies in immunoassays and new generations of lateral-flow assays. Novel signal amplification technologies based either on new classes of biofunctional nanocrystals consisting of releasable fluorophores or on aggregation-induced emission (AIE) can improve the sensitivity and the limits of detection in immunoassays. A bio-barcode assay also allows signal amplification by utilizing antibody-coated magnetic beads to concentrate the analytes and antibody-coated gold nanoparticle probes to carry with a large number of oligonucleotides. These innovative technologies boost the development of immunoassays. Growth in rapid immunoassay is fueled by the increasing number of diabetics, the globalization of infectious diseases and the surge in cardiovascular and other chronic diseases as well as other chronic conditions. Rapid, near patient, decentralized, point-of-care (POC) tests are emerging as a tool for more efficient diagnosis and patient evaluation. Technological innovations in lateral-flow assays have enabled a move to bring testing closer to the patient. A novel "digital-style" lateral-flow assay provides semi-quantitative results by simply counting the number of red lines in the test without any expensive reading instrument. An immuno-threshold-based assay can give a signal directly proportional to the concentration of a hapten to prevent confusion on interpretation of the test results. In addition, POC tests become more meaningful to healthcare professionals by combining the benefits of new technologies to provide quantitative results. A molecular compact disc provides a high-resolution imaging capability that can identify and quantify many different antigens simultaneously in highly complex immunoassays. Further advances in immunoassays will bring diagnostic testing even closer to the patient, and can help physicians to monitor diseases that require immediate test results, thereby enhancing the quality of patient care. PMID:17874052

Chan, Cangel Pui-yee; Cheung, Yiu-chi; Renneberg, Reinhard; Seydack, Matthias

2008-01-01

203

Metabolism of IgM in the tropical splenomegaly syndrome.  

PubMed

IgM metabolism was studied in five patients with the tropical splenomegaly syndrome and in five controls. The half-life and turnover time of I125 IgM was similar in the two groups. We conclude that the raised serum IgM levels found in patients with the tropical splenomegaly syndrome are not due to decreased IgM catabolism but are due to increased IgM production. PMID:1006767

Fakunle, Y M; Greenwood, B M

1976-01-01

204

Metal-enhanced immunoassays.  

PubMed

The surface-confined assay format is one of the most convenient detection formats used in many immunoassays. Fluorescence emission from monolayers of dyes requires a strong excitation and good detection system. Such samples are susceptible to artifacts due to background fluorescence from substrates. We demonstrate that using silver nanostructures deposited on the slide substrate can significantly enhance measured fluorescence, reduce unwanted background and increase photostability of the used probes. Using thin layers of polymer doped with fluorescein, we tested two nanostructures--silver island films (SIFs) deposited on glass slides and self-assembled colloidal structures (SACS) deposited on thin silver film. The SACS surfaces show extraordinary fluorescence enhancements: over 100-folds in hot spots. We applied these surfaces for enhanced Alexa488 model immunoassay. PMID:22573442

Gryczynski, Ignacy; Luchowski, Rafal; Matveeva, Evgenia G; Shtoyko, Tanya; Sarkar, Pabak; Borejdo, Julian; Akopova, Irina; Gryczynski, Zygmunt

2012-01-01

205

IgG and IgM autoantibody differences in discoid and systemic lupus patients  

PubMed Central

Systemic lupus (SLE) patients with discoid lupus (DLE) were reported to have milder disease. To test this observation, we employed sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE?SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE?) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE?SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE? (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (ten against nuclear antigens) and four IgM autoantibodies that were differentially expressed (q-value<0.05). DLE?SLE+ subjects had higher IgG autoantibodies against dsDNA, ssDNA, dsRNA, histone H2A and H2B, and SS-A (52 kDa) than all other groups including DLE+SLE+ subjects (p<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (p<0.05). Healthy and DLE+SLE?subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat shock cognate 70, and desmoglein-3 than DLE+SLE+ and DLE?SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE?SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE?SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE?subjects may be non-pathogenic. PMID:22763789

Chong, Benjamin F.; Tseng, Lin-chiang; Lee, Thomas; Vasquez, Rebecca; Li, Quan Z.; Zhang, Song; Karp, David R.; Olsen, Nancy J.; Mohan, Chandra

2012-01-01

206

Replacements for Hydrogen Peroxide for Use With Horseradish Peroxidase in Immunoassay.  

National Technical Information Service (NTIS)

This invention pertains to improving stability of immunoassays by using a more stable component. More specifically, this invention pertains to the use of a stable source of hydrogen peroxide in a peroxidase enzyme assay wherein the source releases hydroge...

D. Kidwell

1991-01-01

207

International standards for IgG and IgM anti-?2glycoprotein antibody measurement.  

PubMed

International standards for anti-beta2 glycoprotein I (anti-?2GPI) testing are needed. We evaluated the suitability of polyclonal/monoclonal candidate reference materials (RM) for the assay. IgG/IgM anti-?2GPI were affinity-purified (AP) from high-positive antiphospholipid syndrome sera and IgG from HCAL clone supernatant. Igs were tested for purity by SDS-PAGE, pooled, concentrated, sterile-filtered and the protein concentration determined. One unit was defined as the binding activity of 1?µg/ml of AP anti-?2GPI Ig. IgG/IgM RM were each assigned a unit value using the respective AP material as a calibrator. Polyclonal/monoclonal RM and 30 samples were evaluated for linearity, unit equivalency and commutability. Polyclonal AP material was assigned a value of 100 U IgG and 15?U IgM anti-?2GPI, respectively. IgG-RM had a value of 270 IgG and the IgM-RM of 220.3 IgM anti-?2GPI U. The linearity (R (2)) of each RM curve for the various assays ranged from 0.96 to 0.99. Commutability samples fit very well within 95% prediction intervals and had excellent correlation when comparing assays. IgG and IgM polyclonal and IgG monoclonal RM displayed excellent linearity and commutability, being good candidates for better standardization of anti-?2GPI immunoassays. PMID:25228737

Willis, R; Grossi, C; Orietta Borghi, M; Martos-Sevilla, G; Zegers, I; Sheldon, J; Meroni, Pl

2014-10-01

208

Low molecular weight IgM in primary biliary cirrhosis.  

PubMed Central

Low molecular weight IgM is the monomeric subunit of pentameric IgM and is not generally found in the blood of healthy individuals. Using a sensitive immunoblotting technique, low molecular weight IgM was detected in all 17 patients with primary biliary cirrhosis and constituted up to 5% of the total circulating IgM. This low molecular weight IgM moiety correlated significantly with total IgM (p less than 0.01) but not with the specific biliary cirrhosis mitochondrial autoantibody anti-M2. Furthermore it was not possible to show that a partially purified sample of low molecular weight IgM contained M2 binding activity. Mitogen stimulated peripheral blood mononuclear cells from two of four patients were observed to secrete low molecular weight IgM in vitro, a finding seen in only one of six healthy subjects. Immunoblot analysis of patients sera revealed the presence of other oligomers of IgM in addition to low molecular weight IgM. In conclusion this study suggests that during the enhanced IgM synthesis observed in primary biliary cirrhosis a defect occurs in the assembly of the IgM pentamer with release of monomeric and oligomeric IgM into the circulation. The pathogenic significance of these circulating low molecular weight IgM species is unknown. Images Figure 1 PMID:2318435

Roberts-Thomson, P J; Shepherd, K

1990-01-01

209

Sequential injection immunoassay for environmental measurements.  

PubMed

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

Soh, Nobuaki; Tanaka, Mayumi; Hirakawa, Koji; Zhang, RuiQi; Nakajima, Hizuru; Nakano, Koji; Imato, Toshihiko

2011-01-01

210

Technical and clinical comparison of two fully automated methods for the immunoassay of CA 125 in serum  

Microsoft Academic Search

The sensitivity and precision of two fully automated enzyme immunoassays, a chemiluminescent enzyme immunoassay (CLEIA) and an enzyme-linked immunosorbent assay (ELISA), for the determination of the ovarian carcinoma antigen CA 125 were evaluated by comparison with an immunoradiometric assay (IRMA). Sera were obtained from patients with ovarian carcinoma (N=28 before treatment and N=24 after treatment), digestive system cancer (N=21 before

Guifeng Yan; Huangxian Ju; Zhichao Liang; Tingqing Zhang

1999-01-01

211

IgM nephropathy; time to act.  

PubMed

Implication for health policy/practice/research/medical education: Much has been published on the epidemiology and clinicopathological characteristics of IgM nephropathy, but there is little information on the etiology,pathogenesis and specific therapy of the disease. Controversy still shrouds the definition and nosologic status of the disease. Well-coordinated and concerted international efforts and collaboration between researchers in the developing and developed countries are needed to make further progress on the above aspects of the disease. PMID:24644539

Mubarak, Muhammed

2014-01-01

212

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.  

PubMed

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection. PMID:10424647

Graham, D A; Foster, J C; German, A; McLaren, I E; Adair, B M; Merza, M

1999-07-01

213

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

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

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

1993-01-01

214

A rapid lateral flow immunoassay for the detection of fungal alpha-amylase at the workplace.  

PubMed

Fungal alpha-amylase is a flour supplement which is added to improve the quality of bakery products. Various studies have shown that exposure to this enzyme is an important risk factor for the development of bakers' allergy and this allergy is reported to be one of the most frequent causes of occupational asthma. A rapid assay was developed to monitor exposure to occupational allergens directly at the workplace. The sensitivity of the developed assay is 0.32 ng amylase mL(-1) in a buffer system with the commercially available alpha-amylase preparation Fungamyl 1600S as the standard. Initial validation tests (n = 33) were performed with airborne and settled dust from an industrial bakery. The new lateral flow immunoassay detected amylase in 22 of the 26 samples regarded as positive in an enzyme immunoassay, and was negative for all seven enzyme immunoassay-negative samples, while the four lateral flow immunoassay-negative/enzyme immunoassay-positive samples all had levels below 2 ng mL(-1). The sensitivity of 2 ng mL(-1) of the amylase lateral flow immunoassay is sufficient for first screening purposes and, therefore, this simple and rapid assay may allow direct on-site demonstration of work-related hazards of bio-allergen exposure. This would be particularly useful in occupational hygiene practice, especially in traditional or small-scale bakeries which lack the technological skills for testing the exposure to respiratory allergens. PMID:16951754

Koets, Marjo; Sander, Ingrid; Bogdanovic, Jelena; Doekes, Gert; van Amerongen, Aart

2006-09-01

215

Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons  

SciTech Connect

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´- tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

2007-07-01

216

Evaluation of three methods to measure anti-Brucella IgM antibodies and interference of IgA in the interpretation of mercaptan-based tests.  

PubMed

The results of a dipstick assay for the detection of immunoglobulin M (IgM) to Brucella smooth lipopolysaccharide (S-LPS) correlated with those of an enzyme-linked immunosorbent assay (ELISA) for IgM and of the serum agglutination test (SAT) performed with and without dithiothreitol. Two sera which were dithiothreitol-sensitive and were dipstick negative were shown to contain specific IgA. The dipstick assay is recommended as a simple method for detecting specific IgM antibodies in acute-phase brucellosis patients. PMID:11478668

Marrodan, T; Nenova-Poliakova, R; Rubio, M; Ariza, J; Clavijo, E; Smits, H L; Diaz, R

2001-08-01

217

Comparison of the Brucella Standard Agglutination Test with the ELISA IgG and IgM in patients with Brucella bacteremia  

Microsoft Academic Search

The presumptive diagnosis of Brucellosis is based on a high or rising antibody titer measured by the Brucella Standard Agglutination Test (SAT). This tests does not discriminate between the immunoglobulin classes (IgG and IgM). The purpose of this study was to compare the diagnostic value of SAT with Brucella Enzyme Linked Immunosorbent Assay (ELISA) IgG and IgM tests in patients

Z. A. Memish; M. Almuneef; M. W. Mah; L. A. Qassem; A. O. Osoba

2002-01-01

218

Monomeric (7S) IgM in chronic liver disease.  

PubMed

Monomeric (7S) IgM was detected by polyacrylamide/agarose gell immunodiffusion and Sephadex G200 gel filtration in 33% of sera from patients with primary biliary cirrhosis (PBC) and 5% of patients with HBsAg-negative chronic active liver disease (CALD). It was not found in the sera of patients with extrahepatic cholestasis, alcoholic liver disease (ALD), HBsAg-positive CALD and normal control subjects. In the PBC group the presence of 7S IgM was associated with significantly higher IgM concentrations and Clq binding activity (P less than 0.001; P less than 0.01 respectively). The antibody specificity of the 7S IgM is unknown. Its presence in patients with high serum IgM concentrations probably reflects failure of complete polymerisation of 7S IgM because of an increased rate of synthesis of the protein. The association of the presence of 7S IgM and high total IgM with immune complexes suggests that the increased rate of synthesis of IgM and formation of immune complexes probably are a result of the same antigenic (or mitogenic) stimulus. PMID:118836

Fakunle, Y M; Aranguibel, F; de Villiers, D; Thomas, H C; Sherlock, S

1979-11-01

219

Monomeric (7S) IgM in chronic liver disease.  

PubMed Central

Monomeric (7S) IgM was detected by polyacrylamide/agarose gell immunodiffusion and Sephadex G200 gel filtration in 33% of sera from patients with primary biliary cirrhosis (PBC) and 5% of patients with HBsAg-negative chronic active liver disease (CALD). It was not found in the sera of patients with extrahepatic cholestasis, alcoholic liver disease (ALD), HBsAg-positive CALD and normal control subjects. In the PBC group the presence of 7S IgM was associated with significantly higher IgM concentrations and Clq binding activity (P less than 0.001; P less than 0.01 respectively). The antibody specificity of the 7S IgM is unknown. Its presence in patients with high serum IgM concentrations probably reflects failure of complete polymerisation of 7S IgM because of an increased rate of synthesis of the protein. The association of the presence of 7S IgM and high total IgM with immune complexes suggests that the increased rate of synthesis of IgM and formation of immune complexes probably are a result of the same antigenic (or mitogenic) stimulus. PMID:118836

Fakunle, Y M; Aranguibel, F; de Villiers, D; Thomas, H C; Sherlock, S

1979-01-01

220

Variability in Telavancin Cross-Reactivity among Vancomycin Immunoassays.  

PubMed

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

McConeghy, Kevin W; Liao, Siyun; Clark, Douglas; Worboys, Philip; Barriere, Steven L; Rodvold, Keith A

2014-12-01

221

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...

222

Field Analytic Technologies: Immunoassay and Enzymatic Assays  

NSDL National Science Digital Library

This site features a discussion of immunoassays in the context of testing for environmental contaminants, including a fairly detailed explanation of how immunoassays are conducted and advantages and limitations in the analysis of environmental problems. There is also a discussion of analytical concerns - interferences, limits of detection, accuracy and precision, calibrating immunoassays, etc.

2011-06-01

223

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.  

PubMed

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?

Teste, Bruno; Kanoufi, Frédéric; Descroix, Stéphanie; Poncet, Pascal; Georgelin, Thomas; Siaugue, Jean-Michel; Petr, Jan; Varenne, Anne; Hennion, Marie-Claire

2011-07-01

224

IgG, IgM, and IgA antinuclear antibodies in discoid and systemic lupus erythematosus patients.  

PubMed

IgG antinuclear antibodies (ANAs) are elevated in patients with systemic lupus erythematosus (SLE) compared with patients with discoid lupus erythematosus (DLE). To provide an expanded immunologic view of circulating ANAs in lupus patients, we compared the expressions of IgG, IgM, and IgA ANAs in DLE and SLE patients. In this cross-sectional study, sera from age-, gender-, and ethnic-matched SLE (N = 35), DLE (N = 23), and normal patients (N = 22) were tested for IgG, IgM, and IgA ANAs using enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence (IIF) with monkey esophagus as substrate. ELISAs showed elevated levels of IgG ANA, IgM ANA, and IgG/IgM ANA ratios in SLE patients compared with DLE and normal patients. IgA ANA expression was higher in SLE and DLE patients versus normal patients. IIF studies showed higher percentages of patients positive for IgG, IgM, and IgA ANAs in the SLE group. Higher IgG/IgM ANA ratios in SLE than DLE show enhanced class-switching and a more sustained humoral response in SLE. They also suggest a potential connection of IgM ANAs with disease containment. PMID:24741342

Jost, Sheridan A; Tseng, Lin-Chiang; Matthews, Loderick A; Vasquez, Rebecca; Zhang, Song; Yancey, Kim B; Chong, Benjamin F

2014-01-01

225

Environmental Chemistry: The Immunoassay Option  

Microsoft Academic Search

Scientific awareness and public concern about the state of our environment have led to unprecedented demands on analytical laboratories. There is now much interest in strategies that will help to lower costs and improve efficiency. Immunoassay (IA) techniques, which are widely used in clinical chemistry, could play a key role in the laboratory of the future. IA screening techniques for

J. P. Sherry; Raymond E. Clement

1992-01-01

226

ON SITE SCREENING: APPLICATION OF NOVEL ACRIDAN ESTERS AS HRP DETECTION REAGENT IN IMMUNOASSAY FOR THE DETECTION OF BETA AGONISTS IN BOVINE URINE  

Microsoft Academic Search

A new enzyme immunoassay is developed capable of detecting clenbuterol at the sub ppb level in veal calf urine using HRP as the label and the new acridan ester GZ-11 as the signal reagent. The immunoassay can be performed in microtiterplates with the signal being detected either in a plate luminometer or on instant film.

Gijsbert Zomer; Nicolaas-Jurgen van Belle

227

IGM heating in fossil galaxy groups  

NASA Astrophysics Data System (ADS)

We study intergalactic medium (IGM) heating in a sample of five fossil galaxy groups by using their radio properties at 610 MHz and 1.4 GHz. The power by radio jets introducing mechanical heating for the sampled objects is not sufficient enough to suppress the cooling flow. Therefore, we discussed shock-, vortex heating, and conduction as alternative heating processes. Further, the 1.4 GHz and 610 MHz radio luminosities of fossil groups are compared to a sample of normal galaxy groups of the same brightest group galaxies (BGGs), stellar mass, and total group stellar mass, quantified using the K-band luminosity. It appears that the fossil BGGs are under luminous at 1.4 GHz and 610 MHz for a given BGG stellar mass and luminosity, in comparison to a general population of the groups. In addition, we explore how the bolometric radio luminosity of fossil sample depends on clusters and groups characteristics. Using the HIghest X-ray FLUx Galaxy Cluster Sample (HIFLUGCS) as a control sample we found that the large-scale behaviours of fossil galaxy groups are consistent with their relaxed and virialized nature.

Miraghaei, H.; Khosroshahi, H. G.; Klöckner, H.-R.; Ponman, T. J.; Jetha, N. N.; Raychaudhury, S.

2014-10-01

228

IGM Heating in Fossil Galaxy Groups  

E-print Network

We study intergalactic medium (IGM) heating in a sample of five fossil galaxy groups by using their radio properties at 610 MHz and 1.4 GHz. The power by radio jets introducing mechanical heating for the sampled objects is not sufficient enough to suppress the cooling flow. Therefore, we discussed shock-, vortex heating, and conduction as alternative heating processes. Further, the 1.4 GHz and 610 MHz radio luminosities of fossil groups are compared to a sample of normal galaxy groups of the same radio brightest (BGGs), stellar mass, and total group stellar mass, quantified using the $K$-band luminosity. It appears that the fossil BGGs are under luminous at 1.4 GHz and 610 MHz for a given BGG stellar mass and luminosity, in comparison to a general population of the groups. In addition, we explore how the bolometric radio luminosity of fossil sample depends on clusters and groups characteristics. Using the HIghest X-ray FLUx Galaxy Cluster Sample (HIFLUGCS) as a control sample we found that the large-scale beh...

Miraghaei, H; Klöckner, H -R; Ponman, T J; Jetha, N N; Raychaudhury, S

2014-01-01

229

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

Microsoft Academic Search

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

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

1999-01-01

230

A homogeneous chemiluminescent immunoassay method.  

PubMed

A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte. PMID:23477541

Akhavan-Tafti, Hashem; Binger, Dean G; Blackwood, John J; Chen, Ying; Creager, Richard S; de Silva, Renuka; Eickholt, Robert A; Gaibor, Jose E; Handley, Richard S; Kapsner, Kenneth P; Lopac, Senja K; Mazelis, Michael E; McLernon, Terri L; Mendoza, James D; Odegaard, Bruce H; Reddy, Sarada G; Salvati, Michael; Schoenfelner, Barry A; Shapir, Nir; Shelly, Katherine R; Todtleben, Jeff C; Wang, Guoping; Xie, Wenhua

2013-03-20

231

The comparability of different neuron-specific enolase immunoassays and its impact on external quality assessment system  

Microsoft Academic Search

SUMMARY Objective: Long-term external quality assessments suggest that the individual results of different immunoassays are often not comparable. Our goal was to assess the possible sources of these differences. Methods: The paper is based on the results of analyses using seven different immunoassays: DELFIA (PerkinElmer), Elecsys 2010 (Roche), Kryptor (B.R.A.H.M.S.), the enzyme-linked immunosorbent assay DRG and three methods based on

232

A New Simple Immunoassay for Detecting Helicobacter pylori Infection: Antigen in Stool Specimens  

Microsoft Academic Search

Background\\/Aim: Several diagnostic tests are available for evaluating Helicobacter pylori (Hp) infection: histological examination, culture of gastric biopsy specimens, rapid urease test, urea breath test and serology. A recently marketed direct enzyme immunoassay (HpSA) detects Hp antigen in stool samples. The aim of our study was to evaluate overall diagnostic sensitivity, specificity and positive and negative predictive values of this

Lorella Fanti; Gianni Mezzi; Annalisa Cavallero; Giampietro Gesu; Claudio Bonato; Enzo Masci

1999-01-01

233

Analytica Chimica Acta 399 (1999) 151160 Microplate immunoassay technique using polyelectrolyte carriers  

E-print Network

for the acceleration of microplate enzyme immunoassays (ELISAs). The proposed assay technique includes (1) carrying out in solution [3]. Therefore, techniques that carry out the immune interaction in solution with the subsequent­6]. However, such assays are practical only in combi- nation with additional devices for separation

Hammock, Bruce D.

234

Comparison of immunoassay field tests and laboratory results for PCB, PAH, BTEX, and mercury contaminated soils  

Microsoft Academic Search

Immunoassay tests were used as in situ field screening tools for simultaneous assessment and remediation of soil contaminated with mercury and organics (polychlorinated biphenyls (PCB), polyaromatic hydrocarbons (PAH), and BTEX). Soil samples from approximately 200 sites including metering and compressor stations were investigated along gas pipelines. The suspected contamination originated from formerly used mercury manometers and pipeline liquids. An enzyme-linked

Hammes

1995-01-01

235

Pr eparation of horseradish peroxidase-carbamide and its use in hapten immunoassays  

Microsoft Academic Search

Summary Preparation of horseradish peroxidase (HRP) carbamide that is HRP linked to urea (HRP-carbamide\\/HRP-U) is demonstrated and its potential application in the development of enzyme immunoassays (EIAs) for haptens is described. In this new strategy, the lysine residues of HRP were acylated and then acylated HRP was activated to create highly reactive functional groups by periodate oxidation of its carbohydrate

T. G. Shrivastav; S. Nara; V. Tripathi; Z. Sayeed; K. Rangari; S. K. Chaube; H. Singh

236

Development of immunoassays for detecting clothianidin residue in agricultural products.  

PubMed

Two enzyme-linked immunosorbent assays (ELISAs) based on polyclonal antibodies (PcAbs) for clothianidin are described: colorimetric detection format (ELISA) and pattern of chemiluminescent assay (CLEIA). Clothianidin hapten was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. Anticlothianidin PcAbs were obtained from immunized New Zealand white rabbits. Under optimal conditions, the half-maximal inhibition concentration (IC??) and the limit of detection (LOD, IC??) of clothianidin were 0.046 and 0.0028 mg/L for the ELISA and 0.015 and 0.0014 mg/L for the CLEIA, respectively. There were no obvious cross-reactivities of the antibodies with its analogues except for dinotefuran. Recoveries of 76.4-116.4% for the immunoassays were achieved from spiked samples. The results of immunoassays for the spiked and authentic samples were largely consistent with gas chromatography. Therefore, the proposed immunoassays would be convenient and satisfactory analytical methods for the monitoring of clothianidin in agricultural products. PMID:23527939

Li, Ming; Sheng, Enze; Cong, Lujing; Wang, Minghua

2013-04-17

237

Isotope-labeled immunoassays without radiation waste  

E-print Network

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

Hammock, Bruce D.

238

Electrokinetic Microstrirring to Enhance Immunoassays  

NASA Astrophysics Data System (ADS)

Electrokinetic microstirring is used to improve the sensitivity of microfluidic heterogeneous immuno-sensors by enhancing the transport in diffusion-limited reactions. The AC electrokinetic force, Electrothermal Flow, is exploited to create a circular stirring fluid motion, thereby providing more binding opportunities between suspended and wall-immobilized molecules. This process can significantly reduce test times, important for both field-portable biosensors and for lab-based assays. A 2-D numerical simulation model is used to predict the effect of electrothermal flow on a heterogeneous immunoassay resulting from an AC potential applied to two parallel electrodes. The binding is increased by a factor of 7 for an applied voltage of 10 Vrms. The effect was investigated experimentally using a high affinity biotin-streptavidin reaction. Microstirred reaction rates were compared with passive reactions. The measurements show on average an order of magnitude increase in binding between immobilized biotin and fluorescently-labeled streptavidin after 5 minutes. Therefore, this technique shows significant promise for reducing incubation time and enhancing the sensitivity of immunoassays.

Feldman, Hope; Sigurdson, Marin; Meinhart, Carl

2006-11-01

239

Environmental mercury measurement by immunoassay  

SciTech Connect

Immunochemical-based analytical methods are commonly used in the medical diagnostic field, but only recently have they been adapted for field-portable environmental applications. BioNebraska has developed an immunoassay, based upon a novel monoclonal antibody to mercuric ions, for the detection of mercury in environmental samples. The user-friendly BiMelyze Mercury Tube Immunoassay generates semi-quantitative results rapidly and economically relative to traditional analytical methods. In this presentation the authors will demonstrate the use of this method with environmental matrices and discuss ongoing in-house and independent field results. Sample preparation and analysis can be completed in the field for numerous samples in less than 40 minutes. Mercury is first extracted from the sample by digestion using a separate kit available from Bio-Nebraska. The inherent limit of detection for mercuric ions in aqueous samples is 0.25 ppb and 0.5 ppm for soils. The method is highly selective for mercury with essentially no interference by other metals or matrices. Thus, the assay is well-suited for low-cost, real-time, user friendly field screening of mercury in soils, sediment and water producing results that correlate well with traditional analytical methods.

Schweitzer, C.; Carlson, L.; Holmquist, B.; Riddell, M.; Wylie, D. [BioNebraska, Inc., Lincoln, NE (United States)

1995-12-31

240

Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation.  

PubMed

Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, ?40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by high-throughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM(+) B cells allowed us to relate unmutated ancestor clones in blood to hypermutated offspring clones in CSF. Unexpectedly, however, we found no evidence for intrathecal isotype switching from IgM to IgG. Our data suggest that the intrathecal milieu sustains a germinal centre-like reaction with clonal expansion and extensive accumulation of somatic hypermutation in IgM-producing B cells. PMID:25060097

Beltrán, Eduardo; Obermeier, Birgit; Moser, Markus; Coret, Francisco; Simó-Castelló, María; Boscá, Isabel; Pérez-Miralles, Francisco; Villar, Luisa M; Senel, Makbule; Tumani, Hayrettin; Hohlfeld, Reinhard; Casanova, Bonaventura; Dornmair, Klaus

2014-10-01

241

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

PubMed

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

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

242

IgM response and resistance to ascites tumor growth  

Microsoft Academic Search

Antibody response and protection against Ehrlich ascites tumor (EAT) was studied in eight EAT-immunized strains of mice (AL\\/N, BALB\\/C, C57BL\\/6J, F1(C57BL\\/6×BALB\\/C), C57BL\\/10J, B10.BR, CBA\\/Ca, SW). The results showed a close association between IgM response and resistance to subsequent tumor challenge. Thus, protection was only achieved in those animals giving a measurable IgM response against EAT cell surface antigens, i.e., all

Jose L. Subiza; Javier Coll; Rita Alvarez; Manuel Valdivieso; Emilio G. de la Concha

1987-01-01

243

Enterovirus type 70-neutralizing IgM in animal sera.  

PubMed

The neutralizing activity against human enterovirus type 70 was found in serum samples from normal cattle, horses, sheep, goats, swine and chickens raised in Japan. The frequency was variable depending upon animal species and the year of bleeding. The neutralizing activity in bovine sera was shown to reside in IgM by sucrose gradient centrifugation and immune gel electrophoresis. These findings suggested that the neutralizing substance in domestic animal sera is the antibody of IgM class elicited by unidentified viruses antigenically related to human enterovirus type 70. PMID:6287063

Sasagawa, A; Miyamura, K; Kono, R

1982-04-01

244

The development of immunoassays for detection of chemical warfare agents  

SciTech Connect

With the advent of enzyme linked immunoabsorbant assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were reported as capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detecting soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition, these antibodies were highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies reported an extension of the level of sensitivity to -80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These antibodies offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances.

Lenz, D.E. [Army Medical Research Inst. of Chemical Defense, Aberdeen Proving Ground, MD (United States)

1995-06-01

245

Serologic Cross-Reactivity of Human IgM and IgG Antibodies to Five Species of Ebola Virus  

PubMed Central

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30–45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n?=?24), Gulu, Uganda (n?=?20), Bundibugyo, Uganda (n?=?33), and the Philippines (n?=?18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV. PMID:21666792

MacNeil, Adam; Reed, Zachary; Rollin, Pierre E.

2011-01-01

246

Application of antibody and fluorophore-derivatized liposomes to heterogeneous immunoassays for d-dimer.  

PubMed

Small unilamellar liposomes comprised of cholesterol and phospholipids, in which one of the lipids is labeled with a fluorophore, have been covalently functionalized with antibodies. The liposomes were conjugated with thousands of fluorescein molecules and 10-20 monoclonal antibodies per liposome. These bifunctional liposomes were used in a direct (sandwich-type) immunoassay for the detection of thromboembolic disorders by assaying for d-dimer. D-dimer is the final and the smallest proteolytic product in the degradation of cross-linked fibrin by the plasma protein plasmin. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate. The liposomes, by virtue of having thousands of fluorophores coupled to one liposome in contrast to one or a few reporter molecules in the conventional fluor-antibody conjugate, performed better on two counts: (1) they lowered the detection limit by a factor of 120 and (2) they provided a 1 order of magnitude amplification in signal. The minimum detectable concentration (MDC) of d-dimer was 5.6 ng/mL with the liposomal assay as compared to an MDC of 674 ng/mL with conventional fluor-antibody conjugate. The results of fluorescence assays were also compared with the results obtained by Singh et al. (Biotechnol. Prog. 1995, 11, 333-341) in an enzyme immunoassay developed using liposomes. These results demonstrate the potential of liposomes in lowering detection limits and increasing the sensitivity of immunoassays. PMID:8857195

Singh, A K; Kilpatrick, P K; Carbonell, R G

1996-01-01

247

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis  

PubMed Central

Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB. PMID:21966416

Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

2011-01-01

248

THE SUBUNITS IN RABBIT ANTI-FORSSMAN IGM ANTIBODY  

PubMed Central

Rabbit IgM anti-Forssman antibody was highly purified and the subunits obtained on reduction and alkylation were labeled radioactively and isolated by two different and unrelated methods. In both cases the subunits were found to have a molecular weight of about 90,000, based on their behavior on density gradient centrifugation and gel filtration, and evidence is given that they contained one light and one heavy chain. The subunits bound only weakly to sheep erythrocyte stroma, and only half could be shown to possess antigen specific sites. The data are consistent with the concept that each anti-Forssman IgM molecule has five effective binding sites, but it is uncertain whether the ineffectiveness of the remaining five H-L chain pairs is inherent in the structure of the IgM molecule or an artifact due to the isolation procedure. Intact IgM anti-Forssman antibody binds very firmly to structures containing multiple repeating antigen sites, and it appears that this is due to the presence of multiple binding sites on the molecule. PMID:5655105

Frank, Michael M.; Humphrey, John H.

1968-01-01

249

Immunoassay, biosensors and other nonchromatographic methods  

E-print Network

for the detection of products containing genetically modified organisms (GMOs). The advantages of immunoassay preparation r versatility for target analytes r cost effectiveness for large numbers of samples r adaptability

Hammock, Bruce D.

250

Fluorescence Polarization Immunoassay of Mycotoxins: A Review  

PubMed Central

Immunoassays are routinely used in the screening of commodities and foods for fungal toxins (mycotoxins). Demands to increase speed and lower costs have lead to continued improvements in such assays. Because many reported mycotoxins are low molecular weight (below 1 kDa), immunoassays for their detection have generally been constructed in competitive heterogeneous formats. An exception is fluorescence polarization immunoassay (FPIA), a homogeneous format that does not require the separation of bound and free labels (tracer). The potential for rapid, solution phase, immunoassays has been realized in the development of FPIA for many of the major groups of mycotoxins, including aflatoxins, fumonisins, group B trichothecenes (primarily deoxynivalenol), ochratoxin A, and zearalenone. This review describes the basic principles of FPIA and summarizes recent research in this area with regard to mycotoxins. PMID:22069541

Maragos, Chris

2009-01-01

251

Development of Enzyme Immunoassays for the Detection of Pyrethroid Insecticides  

Microsoft Academic Search

Three monoclonal antibodies (mab) for the determination of the pyrethroid insecticide allethrin were generated using a trans-permethric acid-bovine serum albumin conjugate as immunogen. Spleen cells of immunized BALB\\/c mice were fused with myeloma cells applying polyethylene glycol. Hybridoma cells were selected by HAT selection and screened for cell lines producing mab. Three stable cell lines could be isolated for further

Sabine Pullen; Bertold Hock

1995-01-01

252

Performance of the BioPlex 2200 flow immunoassay in critical cases of serodiagnosis of toxoplasmosis.  

PubMed

The BioPlex 2200 automated analyzer (Bio-Rad Laboratories, Hercules, CA) is a recently developed multiplex analyzer that enables the detection of anti-Toxoplasma, -rubella, and -cytomegalovirus antibodies in the same assay. The aim of this study was to compare this new technology (using the BioPlex 2200 ToRC IgG/IgM kit) in critical cases of serodiagnosis of toxoplasmosis (acute, chronic, or congenital infections and cases with discrepant results) to the technologies used in our routine practice, i.e., the Platelia IgG/IgM enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad Laboratories) and the Toxo-Screen direct agglutination assay (bioMérieux, Lyon, France). Overall, most cases of false-positive/negative results obtained with the Platelia IgG or Toxo-Screen assay were corrected by the BioPlex 2200 ToRC IgG (87.5%). Furthermore, the analysis of 35 sequences of sera showed a trend toward a more rapid decrease of IgM titers by BioPlex 2200 than by Platelia. These results for IgM detection can be explained by a weaker detection of residual IgM. Indeed, among 23 serum samples from patients with probable past infection with long-lasting IgM (Platelia M positive and IgG avidity index, ?0.5), the BioPlex 2200 Toxoplasma IgM assay was positive for only 11 serum samples. In our panel of critical cases comprising 156 serum and 6 cord blood samples from 103 patients with acute, chronic, or congenital infection, the BioPlex 2200 IgG assay was a sensitive (97.8%) and specific (91.3%) method for IgG detection. The high specificity (97.4%) of IgM detection combined with the shorter kinetics of IgM titers may considerably reduce the number of residual IgM detections, thus yielding more precise diagnoses of acute infections. PMID:24477853

Guigue, Nicolas; Menotti, Jean; Hamane, Samia; Derouin, Francis; Garin, Yves Jean-François

2014-04-01

253

Ultrasensitive signal amplified immunoassay of medroxyprogesterone acetate (MPA) using the atomic absorption of silver deposited on the surface of gold nanoparticles  

Microsoft Academic Search

An ultrasensitive determination method was developed to detect the trace medroxyprogesterone acetate (MPA) residues in food. The operation procedure was mostly the same as the traditional enzyme-linked immunosorbent assay method. The gold nanoparticle labelled goat anti-rabbit antibody was used here to take the traditional enzyme labelled antibody. Then the gold labelled antibody was used in the immunoassay as usual. The

Gang Cui; Huaqin Chu; Yongming Hu; Chuanlai Xu

2010-01-01

254

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification  

Microsoft Academic Search

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when

M. Ermolli; A. Prospero; B. Balla; M. Querci; A. Mazzeo; G. Van Den Eede

2006-01-01

255

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

PubMed

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

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

256

Anodic-stripping voltammetric immunoassay for ultrasensitive detection of low-abundance proteins using quantum dot aggregated hollow microspheres.  

PubMed

A new anodic-stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer-by-layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti-human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F(ab) of IgG1 analyte from the sample. For detection, monoclonal anti-human IgG1 (F(c)-specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich-type immunoassay format, subsequent anodic-stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0?fg?mL(-1) to 1.0??g?mL(-1) of IgG1 with a detection limit of 0.1?fg?mL(-1). The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme-linked immunosorbent assay method. The new immunoassay is promising for enzyme-free, and cost-effective analysis of low-abundance biomarkers. PMID:23292875

Zhang, Bing; Tang, Dianping; Goryacheva, Irina Yu; Niessner, Reinhard; Knopp, Dietmar

2013-02-11

257

Liver Abscesses and Hyper IgM Syndrome  

PubMed Central

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

Shah, Ira; Rahangdale, Aarti; Bhatnagar, Sushmita

2013-01-01

258

Recognition of human IgM subgroups by quantitative measurement  

PubMed Central

By means of the single radial immunodiffusion technique it is possible to detect at least two subgroups of human IgM paraproteins, if the square ring diameter is plotted against absolute concentration. These groups give different calibration lines with the same antiserum, even when no qualitative differences can be detected by the double diffusion technique. The possibility of distinguishing these groups is not limited to the use of a single antiserum. The differences between the groups are abolished by reduction of the IgM paraprotein to 7S subunits and therefore seem to be of conformational nature. No identity with other known classifications has yet been found. In one out of two cases investigated differences in quantitative reactivity were found between 19S and >19S components in the same serum. The results suggest that quantitative methods may be used to detect differences in immunoglobulin structure, when qualitative methods fail. They show that different human IgM paraproteins cannot be directly compared quantitatively by the radial immunodiffusion method. PMID:4128890

Klein, F.; De Bruyn, A. M.; Radema, H.

1973-01-01

259

Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles  

Microsoft Academic Search

We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng\\/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma

Shixing Tang; Mahtab Moayeri; Zhaochun Chen; Harri Harma; Jiangqin Zhao; Haijing Hu; Robert H. Purcell; Stephen H. Leppla; Indira K. Hewlett

2009-01-01

260

Evaluation of Commercially Available Diagnostic Tests for the Detection of Dengue Virus NS1 Antigen and Anti-Dengue Virus IgM Antibody.  

PubMed

Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%. PMID:25330157

Hunsperger, Elizabeth A; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L; Artsob, Harvey; Guzman, Maria G; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W; Margolis, Harold S

2014-10-01

261

Evaluation of Commercially Available Diagnostic Tests for the Detection of Dengue Virus NS1 Antigen and Anti-Dengue Virus IgM Antibody  

PubMed Central

Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%. PMID:25330157

Hunsperger, Elizabeth A.; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A.; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L.; Artsob, Harvey; Guzman, Maria G.; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W.; Margolis, Harold S.

2014-01-01

262

Development of an Ultrasensitive Immunoassay for Detecting Tartrazine  

PubMed Central

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

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

2013-01-01

263

Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.  

PubMed

Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels. PMID:24054232

Plana, Emma; Moreno, Maria-José; Montoya, Ángel; Manclús, Juan J

2014-01-15

264

Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine*  

PubMed Central

To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC50) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products. PMID:22302425

Yi, Jian; Meng, Meng; Liu, Zhong-qiu; Zhi, Jin-fang; Zhang, Yuan-yang; Xu, Jing; Wang, Ya-bin; Liu, Jin-ting; Xi, Ri-mo

2012-01-01

265

The development of immunoassays for detection of chemical warfare agents  

SciTech Connect

With the advent of enzyme linked immunoabsorbent assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detection soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition these antibodies were found to be highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies extended the level of sensitivity to {approximately}80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These reagents offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances. More recent efforts have focussed on developing antibodies specific for sulfur mustard a highly reactive vesicating agent.

Lenz, D.E.; Brimfield, A.A.; Cook, L. [Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD (United States)

1996-10-01

266

Miniaturized immunoassays: moving beyond the microplate.  

PubMed

After more than 40 years, immunoassays are still the backbone of protein biomarker analysis in clinical diagnostics and drug development. They have come a long way since their inception, incorporating technical developments including monoclonal antibodies, novel labels and lately microfluidics. A number of microfluidic platforms have been tested, such as centrifugational compact disc assays, lab-on-a-chip, arrays and digital electrochemical assays. This review focuses on commercial applications of microfluidic immunoassays with reference to some applied academic examples of interest. Advantages and disadvantages of the platform technologies are discussed in general. PMID:22250800

Verch, Thorsten; Bakhtiar, Ray

2012-01-01

267

ENZYME: Enzyme Nomenclature Database  

NSDL National Science Digital Library

Recently updated, the ENZYME: Enzyme Nomenclature Database is based mainly on recommendations by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and "describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided." An online user manual describes how to access and use the database, which may be searched by EC number, enzyme class, official description or alternative name(s), chemical compound, or cofactor. Typical returns include Names, Reaction catalyzed, Comments, Human Genetic Diseases, and a host of hyperlinked cross-references. ENZYME is provided by the Swiss Institute of Bioinformatics.

268

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...

269

Immunoassay for Monitoring Environmental and Human Exposure  

E-print Network

such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p-dioxin (PCDDs), polychlorinated dibenzofurans (PCDFsImmunoassay 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

Hammock, Bruce D.

270

Immunoassay procedures for fiber optic sensors  

Microsoft Academic Search

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

Frances S. Ligler

1988-01-01

271

Heavy element enrichment in the IGM at high redshift  

E-print Network

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 $z=1-2.5$.

S. Savaglio

1997-09-16

272

Ly1 B Cells: Functionally Distinct Lymphocytes That Secrete IgM Autoantibodies  

Microsoft Academic Search

Studies presented here introduce another perspective on the mechanisms responsible for IgM autoantibody production. A unique subpopulation of B lymphocytes (Ly-1 B) that concomitantly expresses IgM, IgD, Ia, and Ly-1 membrane glycoproteins is present at higher frequencies in NZB and NZB-related mice. The Ly-1 B subpopulation in these autoimmune animals is responsible for the ``spontaneous'' IgM secretion demonstrated with cultured

Kyoko Hayakawa; Richard R. Hardy; Masaaki Honda; Leonard A. Herzenberg; Alfred D. Steinberg; Leonore A. Herzenberg

1984-01-01

273

IgM nephropathy: timely response to a call for action  

PubMed Central

Immunoglobulin M (IgM) is the largest antibody molecule and is deposited in the glomeruli in a wide variety of both primary and secondary glomerulopathies. The data on its pathogenic role are conflicting till date. A recent study provides evidence for the involvement of natural antibody IgM in fixing and activating complement and causing glomerular injury, proteinuria, and glomerulosclerosis in an animal model. This finding is important in understanding the pathogenesis of the related disorder of IgM nephropathy.

Mubarak, Muhammed; Nasri, Hamid

2014-01-01

274

Low molecular weight IgM and CD5 B lymphocytes in rheumatoid arthritis.  

PubMed Central

OBJECTIVES--To evaluate the role of low molecular weight (LMW) IgM and CD5 B cells in rheumatoid arthritis (RA) and to explore the possibility that LMW IgM is derived selectively from this subset of B cells. METHODS--LMW IgM in sera and culture supernatants was detected by a sensitive immunoblot technique with an enhanced chemiluminescence detection system. CD5 B cells were determined by FACScan cytometry. In vitro studies were established in culture plates containing pokeweed mitogen with or without 2-mercaptoethanol (2-ME). Supernatants were obtained from CD5 positive hybridomas and CD5 negative hybridomas. Other immunological indices were measured by laser nephelometry. RESULTS--Circulating LMW IgM was detected in all rheumatoid patients with significantly higher levels being observed in sero-positive patients. LMW IgM correlated significantly with total IgM and RF. Peripheral blood mononuclear cells (PBMC) from the majority of the patients with RA secreted LMW IgM in vitro as did mononuclear cells from a synovial fluid sample. The addition of low concentrations of 2-ME to the culture medium enhanced the proportions of secreted monomeric IgM. In contrast, PBMC from healthy subjects secreted only trace quantities of LMW IgM. In RA no significant correlations were observed between CD5 B cells and LMW IgM and RF. LMW IgM could be detected in the supernatants from both CD5+ and CD5- B cell lines. Finally, CD5 B cells were not significantly elevated in RA and levels remained constant over time. CONCLUSION--LMW IgM exists in high concentrations in RA sera and synovial fluid. Serum level correlates with RF and IgM. In vitro studies have suggested that the occurrence of LMW IgM may be due to an intrinsic defect(s) in the assembly of the IgM pentameric molecule. LMW IgM is unlikely to be derived solely from CD5 B cells. Images PMID:7518662

Xu, H; Geddes, R; Roberts-Thomson, P J

1994-01-01

275

Determination of naphthalene by competitive fluorescence immunoassay.  

PubMed

A reliable and sensitive competitive fluorescence immunoassay for the quantitative determination of naphthalene (NA) was developed. 2-naphthoxy acetic acid (NAA) was selected as the hapten of naphthalene. Active ester method (AEM) was used to couple the NAA to carrier proteins (bovine serum albumin) to form artificial immune antigen. Male New Zealand white rabbits were immunized with this antigen to obtain polyclonal antibodies, with which, a novel fluorescence immunoassay for detection of NA was described. Under best conditions, NA can be determined in the concentration range of 0.1-100 microg/L with a detection limit of 0.05 microg/L. The cross-reactivities of the anti-NA antibody to seven structurally related compounds were below 15%. Some environmental samples were analyzed with satisfactory results. It shows a good accuracy and suitability to analyze NA in environmental water. PMID:18553145

Zhou, Chun; Wang, Qiong-E; Gao, Shan-Shan; Zhuang, Hui-Sheng

2009-07-01

276

Association between IgM Anti-Herpes Simplex Virus and Plasma Amyloid-Beta Levels  

E-print Network

Association between IgM Anti-Herpes Simplex Virus and Plasma Amyloid-Beta Levels Catherine Fe´art1, University of Lille 2, Lille, France, 8 INSERM, U837, Lille, France Abstract Objective: Herpes simplex virus, et al. (2011) Association between IgM Anti-Herpes Simplex Virus and Plasma Amyloid-Beta Levels. PLo

Paris-Sud XI, Université de

277

Low molecular weight IgM in selective IgA deficiency.  

PubMed Central

Thirty-nine persons with selective IgA deficiency were studied. These comprised 27 subjects found by population screening and 12 by other means. Low molecular weight (LMW) serum IgM was sought in 28 of the 39 persons. Nine of the 28 (32%) had LMW IgM detectable by a sensitive gel filtration technique. Of 17 patients discovered by screening, five (29%) had LMW IgM. In the nine positive persons, LMW IgM constituted up to 17% of the total serum IgM concentration. Eight of the nine IgA deficient persons with LMW IgM, had clinical disease while associated disease in the entire IgA deficient population was less frequent. Serum immune complexes were demonstrated in five of seven subjects with LMW IgM using a C1q-dependent radioimmunoassay; four of these had immune complex associated disorders, three with polyarthritis and one with glomerulonephritis. Because circulating immune complexes are frequently detected in IgA deficient persons without disease, it is proposed that the presence of LMW serum IgM in IgA deficiency may be associated with disease due to the formation of specific pathogenic immune complexes. PMID:7172505

Kwitko, A O; Roberts-Thomson, P J; Shearman, D J

1982-01-01

278

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

NASA Astrophysics Data System (ADS)

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.

Zhao, Haiying; Dou, Xiaoming

2005-01-01

279

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. ...

280

Utility of a novel HCV-NS4 antigen detection immunoassay for monitoring treatment of HCV-infected individuals with pegylated interferon ?-2a  

Microsoft Academic Search

Hepatitis C virus (HCV) infection continues to be an emerging global health concern. With the advent of additional treatment protocols, a simple and reliable assay for changes in HCV load may permit more frequent patient assessment and tailoring of the therapeutic regimen. Recently, we have developed a highly sensitive and specific enzyme immunoassay (EIA) for the detection of an HCV-Nonstructural

Abdelfattah M. Attallah; Gamal E. Shiha; Camelia A. Abdel Malak; Hytham E. Hagras; Waleed S. Abdel-Razik; Hisham Ismail

2004-01-01

281

Pancreatic Enzymes  

MedlinePLUS

Pancreatic enzymes Ver esta página en espańol What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and carbohydrates. A ... into the duodenum, daily. This fluid contains pancreatic enzymes to help with digestion and bicarbonate to neutralize ...

282

Ultrarapid generation of femtoliter microfluidic droplets for single-molecule-counting immunoassays.  

PubMed

We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10(6) per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules. PMID:23805985

Shim, Jung-uk; Ranasinghe, Rohan T; Smith, Clive A; Ibrahim, Shehu M; Hollfelder, Florian; Huck, Wilhelm T S; Klenerman, David; Abell, Chris

2013-07-23

283

Multiplexed electrochemical immunoassay of biomarkers using metal sulfide quantum dot nanolabels and trifunctionalized magnetic beads.  

PubMed

A novel multiplexed stripping voltammetric immunoassay protocol was designed for the simultaneous detection of multiple biomarkers (CA 125, CA 15-3, and CA 19-9 used as models) using PAMAM dendrimer-metal sulfide quantum dot (QD) nanolabels as distinguishable signal tags and trifunctionalized magnetic beads as an immunosensing probe. The probe was prepared by means of co-immobilization of primary monoclonal anti-CA 125, anti-CA 15-3 and anti-CA 19-9 antibodies on a single magnetic bead. The PAMAM dendrimer-metal sulfide QD nanolabels containing CdS, ZnS and PbS were synthesized by using in situ synthesis method, which were utilized for the labeling of polyclonal rabbit anti-CA 125, anti-CA 15-3 and anti-CA 19-9 detection antibodies, respectively. A sandwich-type immunoassay format was adopted for the simultaneous determination of target biomarkers in a low-binding microtiter plate. The subsequent anodic stripping voltammetric analysis of cadmium, zinc, and lead components released by acid from the corresponding QD nanolabels was conducted at an in situ prepared mercury film electrode based on the difference of peak potentials. Experimental results indicated that the multiplexed immunoassay enabled the simultaneous detection of three cancer biomarkers in a single run with wide dynamic ranges of 0.01-50 U mL(-1) and detection limits (LODs) of 0.005 U mL(-1). Intra-assay and inter-assay coefficients of variation (CVs) were less than 7.2% and 10.4%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum specimens between the multiplexed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA). PMID:23500474

Tang, Dianping; Hou, Li; Niessner, Reinhard; Xu, Mingdi; Gao, Zhuangqiang; Knopp, Dietmar

2013-08-15

284

Industrial Fungal Enzymes: An Occupational Allergen Perspective  

PubMed Central

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

Green, Brett J.; Beezhold, Donald H.

2011-01-01

285

The new ParaDIgm: IgM from bench to clinic  

PubMed Central

The inaugural IgM event entitled “The new ParaDIgm: IgM from bench to clinic” brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23–24, 2013 in Frankfurt, Germany. PMID:22864407

Hanala, Sherif

2012-01-01

286

Gliadin Detection in Food by Immunoassay  

NASA Astrophysics Data System (ADS)

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.

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

287

Fluorescent immunoassay visualization of sorbed pollutants  

SciTech Connect

Current methods of detecting sorbed soil pollutants require that the contaminant be extracted from the soil. In an effort to make detection simpler and safer, standard fluorescent immunoassay techniques are being modified to allow fluorescent tags on the pollutant to be viewed and photographed with epifluorescent microscopy. Initial research focuses on detecting chlorinated benzenes on various soil types and developing a technique for tagging these pollutants with appropriate antibodies. This should lead to detection in actual soil cores and a better understanding of how contaminants progress through different soils.

Moore, W.K.; Mossman, D.J. [Univ. of Missouri-Columbia, Kansas City, MO (United States). Dept. of Civil Engineering; Schwab, A.P. [Kansas State Univ., Manhattan, KS (United States). Dept. of Agronomy; Feldbush, T.L. [Northwestern Univ., Evanston, IL (United States)

1994-12-31

288

Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays  

SciTech Connect

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.

Liu, Guodong; Lin, Yuehe

2007-12-15

289

Transcriptional Heterogeneity of IgM+ Cells in Rainbow Trout (Oncorhynchus mykiss) Tissues  

PubMed Central

Two major classes of B lymphocytes have been described to date in rainbow trout: IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcR?. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM+ populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM+ B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity. PMID:24324826

Abos, Beatriz; Castro, Rosario; Pignatelli, Jaime; Luque, Alfonso; Gonzalez, Lucia; Tafalla, Carolina

2013-01-01

290

Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides.  

PubMed

A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides. PMID:12033799

Chin, Tina E; Wong, Rosie B; Pont, Joseph L; Karu, Alexander E

2002-06-01

291

Microfluidic immunoassays as rapid saliva-based clinical diagnostics  

PubMed Central

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

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

2007-01-01

292

Fluorimetric Immunoassay for Multianalysis of Aflatoxins  

PubMed Central

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

2013-01-01

293

Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin.  

PubMed

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L. PMID:8504565

Cook, D B; Self, C H

1993-06-01

294

DEVELOPMENT OF AN ANTIGEN HETEROLOGOUS ENZYME LINKED IMMUNOSORBENT ASSAY FOR MEASUREMENT OF CORTICOSTERONE IN RAT SERUM  

Microsoft Academic Search

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop corticosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against corticosterone-3-carboxymethyloxime-bovine serum albumin (corticosterone-3-CMO-BSA) and corticosterone-21-hemisuccinate-bovine serum albumin (corticosterone-21-HS-BSA). Four horseradish peroxidase (HRP) enzyme conjugates were prepared using two corticosterone derivatives (corticosterone-3-CMO

Tulsidas G. Shrivastav; Shail K. Chaube; Kiran P. Kariya; Kiran Rangari; Rita Singh

2011-01-01

295

Modelling the IGM and the Ly alpha forest at high redshift from the dark matter distribution  

Microsoft Academic Search

A variety of approximate schemes for modelling the low-density intergalactic medium (IGM) in the high-redshift Universe are compared with the results of a large high-resolution hydrodynamical simulation. These schemes use either an analytical description of the dark matter distribution and the IGM or numerical simulations of the dark matter (DM) distributions combined with different approximate relations between dark matter field

M. Viel; S. Matarrese; H. J. Mo; Tom Theuns; M. G. Haehnelt

2002-01-01

296

Application of linear discriminant analysis in performance evaluation of extractable nuclear antigen immunoassay systems in the screening and diagnosis of systemic autoimmune rheumatic diseases.  

PubMed

This study applied a linear discriminant analysis model to evaluate the performance of 2 types of commercially available extractable nuclear antigen (ENA) immunoassays for the screening and diagnosis of systemic autoimmune rheumatic diseases (SARDs) in a large tertiary hospital reference laboratory: (1) an enzyme-linked immunosorbent assay (ELISA) and (2) a multiplex bead-based immunoassay (MPBI). The results of the study showed both ENA immunoassays had comparable sensitivity for the detection of SARDs compared with the antinuclear antigen immunofluorescence (ANA-IF) method (ANA-IF: 85.6%, ENA-ELISA: 91.5%, ENA-MPBI: 83.1%, pairwise comparisons with ANA-IF: P > .05). However, both ENA immunoassays offered improved specificity compared with the ANA-IF (ANA-IF: 24.2%; ENA-ELISA: 39.8%; ENA-MPBI: 53.1%; pairwise comparison with ANA-IF: P < .001). The use of a more specific screening immunoassay with comparable sensitivity to ANA-IF is important in a tertiary hospital with high prevalence of non-SARD immune diseases. Diagnostic performance of the ENA/dsDNA components by the MPBI and ELISA methods did not differ significantly (area under the curve [AUC], 81.0% vs 83.0%, respectively, P > .05), but the key ENA/dsDNA variables contributing to the discriminating power of the assays for the diagnosis of specific SARDs were reagent/method dependent. PMID:23010715

Pi, David; de Badyn, Monika Hudoba; Nimmo, Mike; White, Rick; Pal, Jason; Wong, Patrick; Phoon, Carmen; O'Connor, Deidre; Pi, Steven; Shojania, Kam

2012-10-01

297

Environmental Immunoassays: Alternative Techniques for Soil and Water Analysis  

USGS Publications Warehouse

Analysis of soil and water samples for environmental studies and compliance testing can be formidable, time consuming, and costly. As a consequence, immunochemical techniques have become popular for environmental analysis because they are reliable, rapid, and cost effective. During the past 5 years, the use of immunoassays for environmental monitoring has increased substantially, and their use as an integral analytical tool in many environmental laboratories is now commonplace. This chapter will present the basic concept of immunoassays, recent advances in the development of immunochemical methods, and examples of successful applications of immunoassays in environmental analysis.

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

1996-01-01

298

The utility of immunoassays for urine drug testing.  

PubMed

Substance abuse is a significant problem in the United States, with cocaine, marijuana, alcohol and heroin as the most commonly abused drugs. This article focuses on urine drug testing to evaluate potential drug abuse or overdose in the emergent care setting using qualitative immunoassays. Discussion is included regarding the principles of how to validate qualitative immunoassays; how to decide on appropriate specimen type, test menu and cutoff; the limitations of immunoassays; how to communicate test results to clinicians; and use of urine drug testing at point of care. PMID:22939301

Melanson, Stacy E F

2012-09-01

299

Immunoassay procedures for fiber optic sensors  

NASA Astrophysics Data System (ADS)

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.

Ligler, Frances S.

1988-04-01

300

Detection of Loxosceles venom in lesional hair shafts and skin: application of a specific immunoassay to identify dermonecrotic arachnidism.  

PubMed

Loxosceles spiders, of which the brown recluse is the best known, are indigenous to southcentral and southwestern regions of the United States. Loxosceles spider envenomation frequently results in painful, centrally necrotic, erythematous skin lesions that evolve over 24 to 48 hours and may take several weeks to completely heal. The diagnosis of loxoscelism is typically is based on the presence of the characteristic dermal lesion, because no definitive clinical diagnostic assay exists, and the spider is generally not available for identification. We used a rapid Loxosceles-specific enzyme immunoassay to detect spider venom in a dermal biopsy and hairs plucked from a suspicious skin lesion on the lower extremity of a 52-year-old man. This report indicates that in using a novel Loxosceles-specific immunoassay, venom can be detected in dermonecrotic skin and hair specimens for up to 4 days after envenomation. PMID:10999583

Miller, M J; Gomez, H F; Snider, R J; Stephens, E L; Czop, R M; Warren, J S

2000-09-01

301

Enzyme Reactions  

NSDL National Science Digital Library

This video shows an enzyme reaction lab. The teacher demonstrates how the enzyme, catalase, reacts with hydrogen peroxide (a substrate found in cells). The teacher first demonstrates a normal enzyme reaction. He or she then goes on to show how manipulating temperature and pH will affect the reaction of an enzyme.

School, Minerva D.

2011-10-03

302

The design and applications of nanoparticle coated microspheres in immunoassays.  

PubMed

Nanoparticle coated microspheres are composed of two or more materials with a core/shell structure and exhibit unique abilities that allow amplification of trace targets in immunoassays. The preparation of nanoparticle coated microspheres can be accomplished using three main strategies: (1) in-situ, (2) ex-situ, and (3) hollow sphere methods. Antibodies or biomolecules can be immobilized on the surface of nanoparticle coated microspheres or hollow spheres to carry out detection of targets using surface-enhanced resonance spectroscopy (SERS), fluorescence, electrochemistry, and many others. Using these particles as antibody carriers in SERS-based immunoassays, broad dynamic range and low detection limits, and improved selectivity can be realized. By assistance of nanoparticle coated microspheres in immunoassays, improved sensitivity and selectivity has been realized. In this review, nanoparticle coated microsphere generation, recent applications, and future potential with respect to immunoassays are discussed. PMID:24730268

Lin, Huei-Shian; Carey, James R

2014-01-01

303

Detection of narcotics with an immunoassay film badge  

SciTech Connect

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.

Lukens, H.R. [Diametrix Detectors, Inc., San Diego, CA (United States)

1993-12-31

304

Multiplexed magnetic microsphere immunoassays for detection of pathogens in foods  

Microsoft Academic Search

Foodstuffs have traditionally been challenging matrices for conducting immunoassays. Proteins, carbohydrates, and other macromolecules\\u000a present in food matrices may interfere with both immunoassays and PCR-based tests, and removal of particulate matter may also\\u000a prove challenging prior to analyses. This has been found true when testing for bacterial contamination of foods using the\\u000a standard polystyrene microspheres utilized with Luminex flow cytometers.

Jason S. Kim; Chris R. Taitt; Frances S. Ligler; George P. Anderson

2010-01-01

305

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.  

PubMed

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 ?M ATP or 10 ?M ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 ?M ADP and 1 ?M AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays. PMID:25372046

Fiene, Amelie; Baqi, Younis; Lecka, Joanna; Sévigny, Jean; Müller, Christa E

2014-12-01

306

Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells?  

PubMed Central

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 ?l of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients. PMID:17116661

Roucairol, Camille; Azoulay, Stephane; Nevers, Marie-Claire; Creminon, Christophe; Lavrut, Thibault; Garraffo, Rodolphe; Grassi, Jacques; Burger, Alain; Duval, Daniele

2007-01-01

307

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

PubMed

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

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

2014-11-01

308

On Modeling and Measuring the Temperature of the z~5 IGM  

E-print Network

The temperature of the low-density intergalactic medium (IGM) at high redshift is sensitive to the timing and nature of hydrogen and HeII reionization, and can be measured from Lyman-alpha forest absorption spectra. Since the memory of intergalactic gas to heating during reionization gradually fades, measurements as close as possible to reionization are desirable. In addition, measuring the IGM temperature at sufficiently high redshifts should help to isolate the effects of hydrogen reionization since HeII reionization starts later, at lower redshift. Motivated by this, we model the IGM temperature at z>5 using semi-numeric models of patchy reionization. We construct mock Lyman-alpha forest spectra from these models and consider their observable implications. We find that the small-scale structure in the Lyman-alpha forest is sensitive to the temperature of the IGM even at redshifts where the average absorption in the forest is as high as 90%. We forecast the accuracy at which the z~5 IGM temperature can be m...

Lidz, Adam

2014-01-01

309

Hapten synthesis and antibody production for the development of a melamine immunoassay.  

PubMed

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC(50) of 70.6 ng mL(-1), a LOD of 2.6 ng mL(-1) and a LOQ of 7.6 ng mL(-1). Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical. PMID:20381695

Lei, Hongtao; Shen, Yudong; Song, Lijun; Yang, Jinyi; Chevallier, Olivier P; Haughey, Simon A; Wang, Hong; Sun, Yuanming; Elliott, Christopher T

2010-04-14

310

Laser-printing of toner-based 96-microzone plates for immunoassays.  

PubMed

This work describes the quick and simple fabrication of toner-based 96-microzone plates by a direct-printing technology. The printer deposits a toner layer (ca. 5 ?m thick) on the polyester surface which acts as a hydrophobic barrier to confine small volumes of sample on test zones (wells). A 96-microzone toner plate was explored to demonstrate its capability of performing enzyme-linked immunosorbent assay (ELISA). The detection of anti-immunoglobulin G (anti-IgG) and immunoglobulin M (IgM) antibodies has been successfully achieved in cell culture and serum samples, respectively. The use of a conventional microplate reader has allowed obtaining a limit of detection of 13 fmol of mouse IgG per zone on printed microplates. The IgM antibody has been detected in a serum sample collected from a patient infected with dengue virus. The detection of a primary infection has been provided by a microplate reader and also by a cell phone camera. Besides the bioanalytical feasibility, toner-based zones have shown good repeatability for inter-zone and intra-plate comparisons. The relative standard deviation (RSD) values for inter-zone (n = 12) and intra-plate (n = 3) comparisons were lower than 6% and 11%, respectively. Furthermore, it was found that the lifetime of each printed microplate depends on the storage temperature. The shelf life for devices stored at 10 °C has been estimated to be ca. four weeks. PMID:23248817

Oliveira, Karoliny Almeida; Rodrigues de Oliveira, Cristina; Antonelli da Silveira, Lucimeire; Coltro, Wendell Karlos Tomazelli

2013-02-21

311

Multiplex chemiluminescent immunoassay for screening of mycotoxins using photonic crystal microsphere suspension array.  

PubMed

A novel multiplex chemiluminescent mycotoxin immunoassay suspension array system was developed by combining the silica photonic crystal microspheres (SPCMs) encoding technique and a chemiluminescent immunoassay (CLIA) method. The SPCMs were used as a carrier of the suspension array and encoded by their reflectance peak positions, which overcome fluorescence photobleaching, and the potential interference between the encoding fluorescence and detection fluorescence. Aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA) artificial antigens were immobilized on the surfaces of SPCMs by using 3-glycidoxypropyltrimethoxysilane as a linker. Horseradish peroxidase (HRP) was used as a labeling enzyme for the secondary antibody in the enzyme-catalyze H2O2-luminol chemiluminescence system. The CLIA detection system was easily integrated with a multifunctional microplate reader and displayed a two to three orders of magnitude dynamic linear detection range from 0.001 to 1, 0.001 to 1, and 0.01 to 1 ng mL(-1) for AFB1, FB1 and OTA with 50% inhibitory concentrations (IC50) of 0.01, 0.036, and 0.04 ng mL(-1), respectively. The recovery rates are in the range of 63.5 to 121.6% for the three mycotoxins in three kinds of spiked cereal samples. The results of detection in 12 naturally contaminated cereal samples were consistent with that of the classic enzyme-linked immunosorbent assay (ELISA) method. This proposed system is simple, rapid, low cost and high throughput for multiplex mycotoxin assay. PMID:24352570

Xu, Kun; Sun, Yue; Li, Wei; Xu, Jie; Cao, Bin; Jiang, Yunkun; Zheng, Tiesong; Li, Jianlin; Pan, Daodong

2014-02-21

312

Enzyme Kinetics  

NSDL National Science Digital Library

This resrouce provides detailed protocols for performing a laboratory exercise in enzyme kinetics. The activity of enzymes are characterized both by reaction rates and the effect of different concentrations of substrates.

Carl Stiefbold (University of Oregon;); Karen Sprague (University of Oregon;); Will Goodwin (University of Oregon;); Sam Donovan (University of Oregon;); Vicki Chandler (University of Oregon;)

1998-01-01

313

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.  

PubMed

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively. PMID:10098683

Graham, D A; Mawhinney, K A; German, A; Foster, J C; Adair, B M; Merza, M

1999-03-01

314

The purification and characterisation of cervine IgM and IgG.  

PubMed

A procedure is described for the isolation of immunoglobulin G (IgG) and immunoglobulin M (IgM) from hyperimmune cervine serum. Hybrids of red deer (Cervus elaphus) and wapiti (Cervus canadensis) were immunised with keyhole limpet hemocyanin (KLH). An immunoglobulin-containing fraction was precipitated from the hyperimmune serum using ammonium sulphate. The antigen-specific immunoglobulins were purified by KLH-conjugated sepharose affinity chromatography and further separated into IgM and IgG by gel-filtration chromatography. Purified immunoglobulin was analysed by polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights and isoelectric points of the composite chains of cervine IgG and IgM are presented. PMID:2075697

Hibma, M H; Griffin, J F

1990-12-01

315

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

PubMed Central

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

2014-01-01

316

Increased urinary IgM excretion in patients with chest pain due to coronary artery disease  

PubMed Central

Background Micro-albuminuria is a recognized predictor of cardiovascular morbidity and mortality in patients with coronary artery disease. We have previously reported, in diabetic and non-diabetic patients, that an increased urinary excretion of IgM is associated with higher cardiovascular mortality. The purpose of this study was to investigate the pattern of urinary IgM excretion in patients with acute coronary syndrome (ACS) and its correlation to cardiovascular outcome. Methods Urine albumin, and IgM to creatinine concentration ratios were determined in 178 consecutive patients presenting with chest pain to the Department of Emergency Medicine (ED) at the University Hospital of Lund. Fifty eight (23 female) patients had ACS, 55 (19 female) patients had stable angina (SA), and 65 (35 female) patients were diagnosed as non-specific chest pain (NS). Results Urine albumin and IgM excretions were significantly higher in patients with ACS (p?=?0.001, and p?=?0.029, respectively) compared to patients with NS-chest pain. During the 2 years follow-up time, 40 (19 female) patients suffered a new major cardiovascular event (ACS, acute heart failure, stroke) and 5 (4 male/1 female) patients died of cardiovascular cause. A high degree of albuminuria and IgM-uria significantly predicted cardiovascular mortality and morbidity (HR?=?2.89, 95% CI: 1.48 - 5.66, p?=?0.002). Microalbuminuric patients (?3?mg/mmol) with high IgM-uria (?0.005?mg/mmol) had a 3-fold higher risk for cardiovascular new events compared to patients with low IgM-uria (RR?=?3.3, 95% CI: 1.1 - 9.9, p?=?0.001). Conclusion In patients with chest pain, an increased urine IgM excretion, is associated with coronary artery disease and long-term cardiovascular complications. Measuring urine IgM concentration could have a clinical value in risk stratification of patients with ACS. PMID:24028208

2013-01-01

317

On-chip immunoassay using surface-enhanced Raman scattering of hollow gold nanospheres.  

PubMed

A surface-enhanced Raman scattering (SERS)-based gradient optofluidic sensor has been developed for a fast and sensitive immunoassay. In this work, a novel microfluidic sensor with functional internal structures has been designed and fabricated. This sensor is composed of three compartments consisting of the gradient channel that serially dilutes the target marker, the injection and mixing area of antibody-conjugated hollow gold nanospheres and magnetic beads, and the trapping area of sandwich immunocomplexes using multiple solenoids. Quantitative analysis of a specific target marker is performed by analyzing its characteristic SERS signals. This SERS-based gradient optofluidic sensor can replace the set of microwells or microtubes used in manual serial dilutions that have been traditionally used in enzyme-linked immunosorbent assay (ELISA)-type assays. The limit of detection for rabbit immunoglobin (IgG) is estimated to be 1-10 ng/mL. This novel SERS-based optofluidic immunoassay system is expected to be a powerful clinical tool for the fast and sensitive medical diagnosis of a disease. PMID:20503972

Chon, Hyangah; Lim, Chaesung; Ha, Seung-Mo; Ahn, Yoomin; Lee, Eun Kyu; Chang, Soo-Ik; Seong, Gi Hun; Choo, Jaebum

2010-06-15

318

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

PubMed Central

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

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

2012-01-01

319

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay  

PubMed Central

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1?20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins. PMID:23049879

Namekar, Madhuri; Kumar, Mukesh; O'Connell, Maile; Nerurkar, Vivek R.

2012-01-01

320

Highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced Raman scattering of hollow gold nanospheres.  

PubMed

A quick and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay technique, using hollow gold nanospheres (HGNs) and magnetic beads, has been developed. Here, HGNs show strong enhancement effects from individual particles because hot spots can be localized on the pinholes in the hollow particle structure. Thus, HGNs can be used for highly reproducible immunoanalysis of cancer markers. Magnetic beads were used as supporting substrates for the formation of the immunocomplex. This SERS-based immunoassay technique overcomes the problem of slow immunoreaction caused by the diffusion-limited kinetics on a solid substrate because all of the reactions occur in solution. For the validation of our SERS immunoassay, a well-known lung cancer marker, carcinoembryonic antigen (CEA), was used as a target marker. According to our experimental results, the limit of detection (LOD) was determined to be 1-10 pg/mL, this value being about 100-1000 times more sensitive than the LOD of enzyme-linked immunosorbent assay. Furthermore, the assay time took less than 1 h, including washing and optical detection steps. PMID:19301845

Chon, Hyangah; Lee, Sangyeop; Son, Sang Wook; Oh, Chil Hwan; Choo, Jaebum

2009-04-15

321

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.  

PubMed Central

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml. Images PMID:1378062

Muller, F; Stiegler, C; Hadding, U

1992-01-01

322

Evaluation of a New Competitive Immunoassay (BioElisa Syphilis) for Screening for Treponema pallidum Antibodies at Various Stages of Syphilis  

Microsoft Academic Search

The BioElisa Syphilis, a new competitive enzyme immunoassay (EIA) for Treponema pallidum whole antigen that uses specific human immunoglobulin G (IgG) antibodies as the competitor, was evaluated for potential use in screening for syphilis at various stages. The results obtained by this competitive EIA were compared with those obtained by the fluorescent treponemal antibody absorption (FTA-abs) test and the T.

ANNE EBEL; LOIC BACHELART; JEAN-MICHEL ALONSO

1998-01-01

323

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

PubMed Central

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

Vojdani, Aristo

2009-01-01

324

Enzyme Informatics  

PubMed Central

Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

2012-01-01

325

Vertical Microreactor Stack Consist of Poly-(Tetrafluoroethylene) Microfluidics for Immunoassay  

NASA Astrophysics Data System (ADS)

This paper reports the first application of high-aspect ratio PTFE microstruscute, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorvent assay. The advantages of PTFE microstructure for the development of lab-on-a-chip due to the extremely high-aspect ratio microstructure and chemical stability of PTFE is discussed. The results of immunoassay shows the successful detection of analyte (mouse IgG) with detection range with 0-100ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the x-ray exposed surface of PTFE microstructue and successful demonstration of antigen-antibody reaction in the PTFE high-aspect ratio microstructure. We also demonstrated the detection of polychlorinated biphenyl (PCB). As the result of demonstration, we successfully detected PCB with ranging analyte concentration of 0.1-10 ng/ml.

Ukita, Yoshiaki; Kondo, Saki; Takeo, Masahiro; Negoro, Seiji; Kataoka, Chiwa; Utsumi, Yuichi

326

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

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

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

2013-01-01

327

Baryonic Content in the Warm-Hot IGM at Low Redshift  

NASA Technical Reports Server (NTRS)

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.

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

2007-01-01

328

Immunoassay panel profile for detecting total PCB content  

SciTech Connect

Immunoassay test kits are being widely used to provide rapid, inexpensive screening of soil samples for the presence of PCBs at or above a given threshold value. Currently available immunoassay methods are sensitive to aroclor preparations that contain the more highly chlorinated PCB congeners. The interpretation of these tests is accomplished by comparison to an appropriate aroclor standard. If PCB contamination at a site has undergone significant changes through weathering or biological degradation, or if contamination has occurred from lower chlorinated species, the relative sensitivity of available test methods is reduced. This paper describes the results of a program to develop and demonstrate an Immunoassay Panel Method that normalizes the recovery of PCBs detected and thereby provides an accurate representation of the total PCB content of a sample. The development and validation of this method, and the associative correlation testing data using laboratory and environmental samples, will be discussed.

Friedman, S.; Allen, R. [EnSys, Inc., Research Triangle Park, NC (United States); Gui, J.; Barren, E.; Berdahl, D. [GE Corporate Research and Development, Schenectady, NY (United States)

1995-12-31

329

Potential applications of immunoassays in studies of flatfish recruitment  

NASA Astrophysics Data System (ADS)

The fisheries recruitment-stock problem, a lack of correlation between measures of reproductive output of the parent stock and recruitment to the fishery, has several potential biotic and abiotic causes. Immunoassays may be useful in examining several aspects of this and several other problems in flatfish ecology: stock identification, parasitism and disease, and trophic interactions. Given stage-specific antisera capable of recognozing antigenic moieties of, for instance, eggs, larvae, or newly-settled juveniles, it is possible to screen stomach contents of many putative predators ( e.g., shrimp or crabs) rapidly for the presence and amounts of platfish prey. This trophic application of immunological methods has great promise for measuring loss of potential recruits to predation. All immunoassays are limited by the quality of antisera used and the researcher's ability to interpret quantitative data in an ecologically meaningful way. Key references for applications of immunoassays in fish-related questions are provided with recommendations for their utilization.

Feller, Robert J.

330

Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 2. Immunoassay evaluation.  

PubMed

Immunoassays for 2,4,6-trichloroanisol (TCA) have been evaluated. The assays were developed after raising antibodies against three different immunizing haptens (1). Lack of reproducibility has been one of the main problems of these assays. Precision was worse on these assays, reaching lower limits of detection. The high lipophilicity of TCA and its, consequently, low water solubility have been found to be the major cause of this problem. A reliable microplate-based enzyme-linked immunosorbent assay (ELISA) has been set after consideration of the TCA physicochemical features and evaluation of important parameters affecting immunoassay performance. The immunoassay uses As78 (developed against hapten B-KLH) and C9-OVA as the coating antigen. The selectivity is high although the brominated analogue 2,4,6-TBA is also recognized. In buffered media containing 7% ethanol, the resulting assay shows a good accuracy with an IC(50) value of 0.53 microgram L(-)(1) and a limit of detection of 0.044 microgram L(-)(1). Red and white wine samples caused important interferences in the immunoassay demonstrating the necessity of a cleanup procedure prior to the ELISA. PMID:12822926

Sanvicens, Nuria; Varela, Begońa; Marco, M Pilar

2003-07-01

331

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

PubMed Central

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

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

2014-01-01

332

?-(1,3)-Glucan Exposure Assessment by Passive Airborne Dust Sampling and New Sensitive Immunoassays?  

PubMed Central

Associations between house dust-associated ?-(1,3)-glucan exposure and airway inflammatory reactions have been reported, while such exposures in early childhood have been suggested to protect against asthma and wheezing. Most epidemiological studies have used reservoir dust samples and an inhibition enzyme immunoassay (EIA) for ?-(1,3)-glucan exposure assessment. The objective of this study was to develop inexpensive but highly sensitive enzyme immunoassays to measure airborne ?-(1,3)-glucans in low-exposure environments, like homes. Specificities of available anti-?-(1,3)-glucan antibodies were defined by direct and inhibition experiments. Three suitable antibody combinations were selected for sandwich EIAs. ?-(1,3)-Glucans in passive airborne dust collected with an electrostatic dust fall collector (EDC) and floor dust from seven homes were measured with the three EIAs. Floor dust samples were additionally analyzed in the inhibition EIA. The sandwich EIAs were sensitive enough for airborne glucan measurement and showed different specificities for commercial glucans, while the ?-(1,3)-glucan levels in house dust samples correlated strongly. The feasibility of measuring glucans in airborne dust with the recently introduced EDC method was further investigated by selecting the most suitable of the three EIAs to measure and compare ?-(1,3)-glucan levels in the EDC and in floor and actively collected airborne dust samples of the previously performed EDC validation study. The EDC ?-(1,3)-glucan levels correlated moderately with ?-(1,3)-glucans in actively collected airborne dust and floor dust samples, while the glucan levels in the airborne dust and floor dust samples did not correlate. The combination of the newly developed ?-(1,3)-glucan sandwich EIA with EDC sampling now allows assessment in large-scale population studies of exposure to airborne ?-(1,3)-glucans in homes or other low-exposure environments. PMID:20038709

Noss, Ilka; Wouters, Inge M.; Bezemer, Gillina; Metwali, Nervana; Sander, Ingrid; Raulf-Heimsoth, Monika; Heederik, Dick J. J.; Thorne, Peter S.; Doekes, Gert

2010-01-01

333

Comparison of ELISA and indirect immunofluorescent antibody assay detecting Coxiella burnetii IgM phase II for the diagnosis of acute Q fever.  

PubMed

A commercially available enzyme-linked immunosorbent assay (ELISA) detecting Coxiella burnetii phase II-specific IgM for the diagnosis of acute Q fever was compared with indirect immunofluorescent antibody assay (IFA). IFA is the current reference method for the detection of antibodies against C. burnetii, but has disadvantages because the judgment of fluorescence is subjective and tiring, and the test is expensive and automation is not possible. To examine whether phase II IgM ELISA could be used as a screening assay for acute Q fever, we compared the sensitivity and specificity of IFA and ELISA. The sensitivity of the IFA and ELISA tests were 100 and 85.7%, respectively, with a specificity of 95.3 and 97.6%, respectively. Because of the high sensitivity and specificity of the ELISA in combination with the practical disadvantages of the IFA, we introduced a new algorithm to screen samples of patients with symptoms of acute Q fever infection. PMID:21997772

Meekelenkamp, J C E; Schneeberger, P M; Wever, P C; Leenders, A C A P

2012-06-01

334

Gender Differences in the Validity of Testosterone Measured in Saliva by Immunoassay  

Microsoft Academic Search

We rigorously evaluated gender differences in the measurement validity of salivary testosterone. Matched serum, saliva, and finger stick blood spot specimens were collected from 40 (20 males) young adults (aged 18–27 years). Saliva was assayed for testosterone by two independent (isotopic and non-isotopic) immunoassay methods. Serum was assayed by commercially available immunoassay kits for free and total testosterone. An immunoassay

Elizabeth A. Shirtcliff; Douglas A. Granger; Andrea Likos

2002-01-01

335

IgM Repertoire Biodiversity is Reduced in HIV-1 Infection and Systemic Lupus Erythematosus  

PubMed Central

Background: HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. Objective: To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 (CDRH3) sequence level. Methods: 454-deep pyrosequencing in combination with a novel analysis pipeline was applied to define populations of IGHM CDRH3 sequences based on absence or presence of somatic hypermutations (SHM) in peripheral blood B cells. Results: HIV or SLE subjects have reduced biodiversity within their IGHM transcriptome compared to healthy subjects, mainly due to a significant decrease in the number of unique combinations of alleles, although recombination machinery was intact. While major differences between sequences without or with SHM occurred among all groups, IGHD and IGHJ allele use, CDRH3 length distribution, or generation of SHM were similar among study cohorts. Antiretroviral therapy failed to normalize IGHM biodiversity in HIV-infected individuals. All subjects had a low frequency of allelic combinations within the IGHM repertoire similar to known broadly neutralizing HIV-1 antibodies. Conclusion: Polyclonal expansion would decrease overall IgM biodiversity independent of other mechanisms for development of the B cell repertoire. Applying deep sequencing as a strategy to follow development of the IgM repertoire in health and disease provides a novel molecular assessment of multiple points along the B cell differentiation pathway that is highly sensitive for detecting perturbations within the repertoire at the population level. PMID:24298273

Yin, Li; Hou, Wei; Liu, Li; Cai, Yunpeng; Wallet, Mark Andrew; Gardner, Brent Paul; Chang, Kaifen; Lowe, Amanda Catherine; Rodriguez, Carina Adriana; Sriaroon, Panida; Farmerie, William George; Sleasman, John William; Goodenow, Maureen Michels

2013-01-01

336

Wavelength-Ratiometric Plasmon Light Scattering-Based Immunoassays  

Microsoft Academic Search

The application of a wavelength-ratiometric plasmon light scattering technique to immunoassays is demonstrated. A model immunoassay\\u000a for anti-immunoglobulin G (IgG), constructed in gold colloid-modified high-throughput screening wells, was monitored by the\\u000a changes in the intensity of scattered light (with transmitted light) from gold colloids as a result of antibody–antibody interactions.\\u000a The quantitative determination of anti-IgG was undertaken by measuring the

Kadir Aslan; Chris D. Geddes

2009-01-01

337

Immunoassay: recent developments and future directions.  

PubMed

All analytical techniques employed in the biological sciences rely on recognition of the shape and structure of molecules of the substance of interest (the analyte). Such molecular recognition and sensing usually relies on the use other molecules possessing a complementary structure, implying a specific lock and key relationship between the two. Antibodies comprise a class of recognition molecules evolved by nature for the purpose of bodily defence, and are clearly of particular utility in this context. However techniques of increasing sophistication (including the techniques of molecular biology) are currently being developed which enable the artificial construction of antibody-like molecules possessing improved molecular recognition properties which can be harnessed for microanalytical purposes. Oligonucleotide probes likewise exhibit the property of binding to complementary nucleotide sequences, and the techniques of, for example, in situ hybridisation therefore share many features with immunoassay techniques. Microanalytical techniques relying on binding reactions between substances possessing complementary lock and key molecular structures are unlikely to be superseded within the foreseeable future, only the labels used to monitor such reactions, and the means of production of "recognition molecules", being subject to further development. Such techniques already enter into all areas of life, including medicine, agriculture, etc, and are likely to increase further in importance with increasing concern regarding chemically complex contaminants in food, the environment, etc. Developments in this field are clearly directed to slightly differing objectives as indicated in this presentation. These include methodological simplification (making the techniques cheaper and more widely available), improvements in sensitivity (to enable the detection and measurement of substances beyond the reach of current methods) and the construction of transducer-based sensor methods (permitting, inter alia, the monitoring of changing analyte concentrations). However the combination of the "ultrasensitivity" of current single analyte assay methods with the ability simultaneously to determine multiple analytes in the same sample represents, in my view, the next major methodological challenge in this field, and--if successfully addressed--will constitute a quantum advance on present analytical methods. Indeed the development of miniaturised multianalyte binding assay techniques may ultimately comes to be seen as analagous to, for example, the introduction of the word processor, and other similar major technological advances of the past decade. PMID:9234310

Ekins, R

1994-04-01

338

AN EVALUATION OF FIVE COMMERCIAL IMMUNOASSAY DATA ANALYSIS SOFTWARE SYSTEMS  

EPA Science Inventory

An evaluation of five commercial software systems used for immunoassay data analysis revealed numerous deficiencies. Often, the utility of statistical output was compromised by poor documentation. Several data sets were run through each system using a four-parameter calibration f...

339

Determination of thevetin B in serum by fluorescence polarization immunoassay.  

PubMed

Thevetin B, a cardiac glycoside of Thevetia neriifolia Juss. seeds, was determined in serum by fluorescence polarization immunoassay. Anti-digitoxin antibody was used, thevetin B genin being structurally identical to digitoxigenin. Cross-reactivity of 94% was found by this method, for concentrations from 5 to 80 ng ml-1. PMID:1420463

Uber-Bucek, E; Hamon, M; Pham Huy, C; Dadoun, H

1992-06-01

340

Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label.  

PubMed

Homogeneous immunoassay is becoming more and more attractive for modern medical diagnosis because it is superior to heterogeneous immunoassay in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin G (IgG) as a model analyte, has been developed. This assay relies upon the catalytic activity of gold nanoparticles (AuNPs) on luminol-AgNO3 chemiluminescence (CL) reaction. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the catalytic activity of AuNPs on luminol-AgNO3 CL reaction is greatly enhanced. Without any separation steps, a CL signal is generated upon addition of a trigger solution, and the CL intensity is directly correlated to the quantity of IgG. The detection limit of IgG was estimated to be as low as 3pg/mL, and the sensitivity was better than that of the reported AuNPs-based CL immunoassay for IgG. PMID:24835732

Luo, Jing; Cui, Xiang; Liu, Wei; Li, Baoxin

2014-10-15

341

A compact immunoassay platform based on a multicapillary glass plate.  

PubMed

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

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

2014-01-01

342

An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification  

PubMed Central

Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability. PMID:22410273

2012-01-01

343

Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label  

NASA Astrophysics Data System (ADS)

Homogeneous immunoassay is becoming more and more attractive for modern medical diagnosis because it is superior to heterogeneous immunoassay in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin G (IgG) as a model analyte, has been developed. This assay relies upon the catalytic activity of gold nanoparticles (AuNPs) on luminol-AgNO3 chemiluminescence (CL) reaction. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the catalytic activity of AuNPs on luminol-AgNO3 CL reaction is greatly enhanced. Without any separation steps, a CL signal is generated upon addition of a trigger solution, and the CL intensity is directly correlated to the quantity of IgG. The detection limit of IgG was estimated to be as low as 3 pg/mL, and the sensitivity was better than that of the reported AuNPs-based CL immunoassay for IgG.

Luo, Jing; Cui, Xiang; Liu, Wei; Li, Baoxin

2014-10-01

344

A Compact Immunoassay Platform Based on a Multicapillary Glass Plate  

PubMed Central

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

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

2014-01-01

345

A Microparticle Enhanced Nephelometric Immunoassay (Nephelia*) Applied to Thymulin Measurement  

Microsoft Academic Search

This article describes a microparticle enhanced nephelometric immunoassay (Nephelia) applied to the quantification of the thymic peptide hormone thymulin. Nephelia uses antibody recognition by the antithymulin antiserum in a competitive reaction between free thymulin and thymulin bound to the microspheres. The binding between microsphere and thymulin is achieved with the aid of a protein carrier. The sensitivity of the competitive

A. Gartner; C. Carles; P. Montagne; M. L. Cuilličre; J. Duheille

1991-01-01

346

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii)  

E-print Network

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used

Ray, David

347

The new ParaDIgm: IgM from bench to clinic: November 15-16, 2011, Frankfurt, Germany.  

PubMed

The inaugural IgM event entitled "The new ParaDIgm: IgM from bench to clinic" brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23-24, 2013 in Frankfurt, Germany. PMID:22864407

Hanala, Sherif

2012-01-01

348

Parasite-specific IgM plays a significant role in the protective immune response to asexual erythrocytic stage Plasmodium chabaudi AS infection.  

PubMed

A comparison of Plasmodium chabaudi AS infection in BALB/c and BALB/c IgM-deficient mice demonstrated a protective role for IgM during infection. IgM-/- mice, unlike microMT mice, display competent B cell humoral immune responses. Increased susceptibility of IgM-/- mice was demonstrated by increased mortality, an advanced ascending infection and higher peak parasitaemia, as well as enhanced anaemia and weight loss compared with wild-type mice. The recrudescent parasitaemias were also higher in the IgM-/- mice. Early specific IgM production in P. chabaudi-infected wild-type mice was followed by IgG1 and IgG2a production, while IgG1 and IgG2a production in IgM-/- mice was preceded by specific IgD production. No protective role for natural IgM against P. chabaudi AS infection was detected as passive transfer of naďve WT serum into IgM-/- mice did not alter the disease outcome or reduce parasite numbers. Passive transfer of WT antiserum, containing predominantly specific IgM, into IgM-/- mice delayed the ascending parasitaemia and reduced mortality. Similarly, coating parasitized red blood cells with WT antiserum, but not IgM-/- antisera, prior to infection also slightly delayed the ascending acute parasitaemia. Specific IgM therefore plays an important role in the limitation of parasite replication during asexual erythrocytic P. chabaudi AS infection. PMID:15987340

Couper, K N; Phillips, R S; Brombacher, F; Alexander, J

2005-05-01

349

Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy.  

PubMed

IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients. In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation. PMID:24529728

Stork, Abraham C J; Jacobs, Bart C; Tio-Gillen, Anne P; Eurelings, Marijke; Jansen, Marc D; van den Berg, Leonard H; Notermans, Nicolette C; van der Pol, W-Ludo

2014-03-15

350

Functional affinity of IgM rheumatoid factor in patients with rheumatoid arthritis and other autoimmune diseases.  

PubMed Central

The functional affinity of IgM rheumatoid factors (RF) was measured in 31 patients with rheumatoid arthritis (RA), 24 with systemic lupus erythematosus (SLE), 13 with Sjögren's syndrome (SS), and in 13 seropositive healthy individuals. The functional affinity of IgM RF from patients with RA was significantly lower than in the other clinical groups studied. In addition, there was a significant inverse correlation between functional affinity and titre of IgM RF in all the groups. These results suggest that the usual mechanisms of affinity based selective pressure (somatic diversification and antigen selection) may operate differently for autoantibodies to serum antigens such as IgG. PMID:3365027

Rath, S; Hogben, D N; Devey, M E

1988-01-01

351

Inadequacy of IgM Antibody Tests for Diagnosis of Rocky Mountain Spotted Fever.  

PubMed

Among 13 suspected Rocky Mountain spotted fever (RMSF) cases identified through an enhanced surveillance program in Tennessee, antibodies to Rickettsia rickettsii were detected in 10 (77%) patients using a standard indirect immunofluorescent antibody (IFA) assay. Immunoglobulin M (IgM) antibodies were observed for 6 of 13 patients (46%) without a corresponding development of IgG, and for 3 of 10 patients (30%) at least 1 year post-onset. However, recent infection with a spotted fever group rickettsiae could not be confirmed for any patient, based on a lack of rising antibody titers in properly timed acute and convalescent serologic specimens, and negative findings by polymerase chain reaction testing. Case definitions used in national surveillance programs lack specificity and may capture cases that do not represent current rickettsial infections. Use of IgM antibodies should be reconsidered as a basis for diagnosis and public health reporting of RMSF and other spotted fever group rickettsiae in the United States. PMID:25092818

McQuiston, Jennifer H; Wiedeman, Caleb; Singleton, Joseph; Carpenter, L Rand; McElroy, Kristina; Mosites, Emily; Chung, Ida; Kato, Cecilia; Morris, Kevin; Moncayo, Abelardo C; Porter, Susan; Dunn, John

2014-10-01

352

Enzyme Action  

NSDL National Science Digital Library

In this activity that can be used as a lab or demonstration, learners use Lactaid® and lactose to demonstrate the concept of enzyme action. Learners test a drop of milk and Lactaid® for the presence of glucose using glucose test paper. Learners also discover the color range of glucose test paper readings. In addition, learners construct paper models to help visualize enzyme action.

Crumlish, Jane

2009-01-01

353

Enzyme Reactions  

NSDL National Science Digital Library

The enzyme reaction rate activity allows students to simulate the effects of variables such as temperature and pH on the reaction rate of the enzyme catalase. This computer simulation is best used after the students have done a wet lab experiment. The value of the simulation is that it requires the students to interpret and analyze the graphical representation of data and it enables the running of mutiple experiments in a short amount of time.

School, Maryland V.

354

Splenectomy Associated Changes in IgM Memory B Cells in an Adult Spleen Registry Cohort  

Microsoft Academic Search

Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited

Paul U. Cameron; Penelope Jones; Malgorzata Gorniak; Kate Dunster; Eldho Paul; Sharon Lewin; Ian Woolley; Denis Spelman

2011-01-01

355

The Evolution of Multiple Isotypic IgM Heavy Chain Genes in the Shark1  

Microsoft Academic Search

The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (VH-D1- D2-JH) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer

Victor Lee; Jing Li Huang; Ming Fai Lui; Karolina Malecek; Yuko Ohta; Arne Mooers; Ellen Hsu

356

Interconversion of different crystal forms of Fabs from human IgM cryoglobulins  

Microsoft Academic Search

In attempts to produce diffraction-quality crystals of Fabs from two human IgM cryoglobulins (Pot and Yvo), we observed unexpected interconversions in crystal morphologies. The Pot Fab crystallized in two forms when polyethylene glycol (PEG) 6,000 was used as the precipitating agent. Broad, relatively short rods with hexagonal cross-sections were our crystals of choice. However, long, thin rods that were often

Paul A. Ramsland; Jadee L. Upshaw; Brandon B. Shultz; Christina R. DeWitt; William F. Chissoe III; Robert L. Raison; Allen B. Edmundson

2001-01-01

357

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons  

NASA Astrophysics Data System (ADS)

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

2013-07-01

358

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons  

PubMed Central

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks. PMID:23881231

Xu, Xiaohua; Wittenberg, Nathan J.; Jordan, Luke R.; Kumar, Shailabh; Watzlawik, Jens O.; Warrington, Arthur E.; Oh, Sang-Hyun; Rodriguez, Moses

2013-01-01

359

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

NASA Astrophysics Data System (ADS)

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.

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

360

A novel acute HIV infection staging system based on 4th generation immunoassay  

PubMed Central

Background Fourth generation (4thG) immunoassay (IA) is becoming the standard HIV screening method but was not available when the Fiebig acute HIV infection (AHI) staging system was proposed. Here we evaluated AHI staging based on a 4thG IA (4thG staging). Findings Screening for AHI was performed in real-time by pooled nucleic acid testing (NAT, n=48,828 samples) and sequential enzyme immunoassay (EIA, n=3,939 samples) identifying 63 subjects with non-reactive 2nd generation EIA (Fiebig stages I (n=25), II (n=7), III (n=29), IV (n=2)). The majority of samples tested (n=53) were subtype CRF_01AE (77%). NAT+ subjects were re-staged into three 4thG stages: stage 1 (n=20; 4th gen EIA-, 3rd gen EIA-), stage 2 (n=12; 4th gen EIA+, 3rd gen EIA-), stage 3 (n=31; 4th gen EIA+, 3rd gen EIA+, Western blot-/indeterminate). 4thG staging distinguishes groups of AHI subjects by time since presumed HIV exposure, pattern of CD8+ T, B and natural killer cell absolute numbers, and HIV RNA and DNA levels. This staging system further stratified Fiebig I subjects: 18 subjects in 4thG stage 1 had lower HIV RNA and DNA levels than 7 subjects in 4thG stage 2. Conclusions Using 4th generation IA as part of AHI staging distinguishes groups of patients by time since exposure to HIV, lymphocyte numbers and HIV viral burden. It identifies two groups of Fiebig stage I subjects who display different levels of HIV RNA and DNA, which may have implication for HIV cure. 4th generation IA should be incorporated into AHI staging systems. PMID:23718762

2013-01-01

361

Multifunctional gold-silica nanostructures for ultrasensitive electrochemical immunoassay of streptomycin residues.  

PubMed

A facile and simple electrochemical immunoassay for ultrasensitive determination of streptomycin residues (STR) in food was designed by using nanogold-assembled mesoporous silica (GMSNs) as bionanolabels on a three-dimensional redox-active organosilica-functionalized sensing interface. To construct such a sensing interface, we initially synthesized organosilica colloids by using wet chemical method, and then utilized the prepared colloidal organosilica nanocomposites for the immobilization of monoclonal anti-STR antibodies on a glassy carbon electrode based on a sol-gel method. The bionanolabels were prepared based on coimmobilization of horseradish peroxidase (HRP) and STR-bovine serum albumin conjugates (STR-BSA) on the GMSNs. With a competitive-type immunoassay format, the assay toward STR analyte was carried out in pH 5.5 acetate acid buffer (ABS) by using redox-active organosilica nanocomposites as electron mediators, biofunctionalized GMSNs as traces, and hydrogen peroxide (H(2)O(2)) as enzyme substrate. Under optimal conditions, the reduction current of the electrochemical immunosensor decreased with the increase in STR level in the sample, and displayed a wide dynamic range of 0.05-50 ng mL(-1) with a low detection limit (LOD) of 5 pg mL(-1) at 3s(B). Intra- and interassay coefficients of variation were less than 8.7 and 9.3% for STR detection, respectively. In addition, the methodology was validated with STR spiked samples including honey, milk, kidney, and muscle, receiving a good correspondence with the results obtained from high-performance liquid chromatography (HPLC). PMID:22059488

Liu, Bingqian; Zhang, Bing; Cui, Yuling; Chen, Huafeng; Gao, Zhuangqiang; Tang, Dianping

2011-12-01

362

Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods.  

PubMed

Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed, in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with ?-glycidoxypropyltrimethoxysilane as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected by chemiluminescent method in the range of 0.5-80 ?g/L and 0.5-80 kU/L with the detection limits of 0.41 ?g/L and 0.36 kU/L at 3? for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1-200 ?g/L and 5-200 kU/L with the detection limits of 0.25 ?g/L and 1.25 kU/L at 3? for CEA and CA19-9, respectively. All these results demonstrated that the present work provided a promising analytical method for tumor markers' analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput detection. PMID:21928078

Wei, Wei; Zhang, Chunyan; Qian, Jing; Liu, Songqin

2011-12-01

363

Development of a microchip Europium nanoparticle immunoassay for sensitive point-of-care HIV detection.  

PubMed

Rapid, sensitive and specific diagnostic assays play an indispensable role in determination of HIV infection stages and evaluation of efficacy of antiretroviral therapy. Recently, our laboratory developed a sensitive Europium nanoparticle-based microtiter-plate immunoassay capable of detecting target analytes at subpicogram per milliliter levels without the use of catalytic enzymes and signal amplification processes. Encouraged by its sensitivity and simplicity, we continued to miniaturize this assay to a microchip platform for the purpose of converting the benchtop assay technique to a point-of-care test. It was found that detection capability of the microchip platform could be readily improved using Europium nanoparticle probes. We were able to routinely detect 5 pg/mL (4.6 attomoles) of HIV-1 p24 antigen at a signal-to-blank ratio of 1.5, a sensitivity level reasonably close to that of microtiter-plate Europium nanoparticle assay. Meanwhile, use of the microchip platform effectively reduced sample/reagent consumption 4.5 fold and shortened total assay time 2 fold in comparison with microtiter plate assays. Complex matrix substance in plasma negatively affected the microchip assays and the effects could be minimized by diluting the samples before loading. With further improvements in sensitivity, reproducibility, usability, assay process simplification, and incorporation of portable time-resolved fluorescence reader, Europium nanoparticle immunoassay technology could be adapted to meet the challenges of point-of-care diagnosis of HIV or other health-threatening pathogens at bedside or in resource-limited settings. PMID:24880655

Liu, Jikun; Du, Bingchen; Zhang, Panhe; Haleyurgirisetty, Mohan; Zhao, Jiangqin; Ragupathy, Viswanath; Lee, Sherwin; DeVoe, Don L; Hewlett, Indira K

2014-11-15

364

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice  

PubMed Central

The IgM antibody response of mice to the streptococcal group A carbohydrate (GAC) was measured. With most strains tested, large amounts of IgM antibody were produced; in AKR mice, over 1% of the total nucleated spleen cells secreted IgM anti-GAC antibody after hyperimmunization. The relative avidity of the antibody was extimated by a modification of the Jerne plaque assay where spleen cells from individual mice were tested against erythrocytes with varying GAC epitope densitymthese studies showed that the earliest, as well as latest, IgM antibodies produced were highly restricted in avidity heterogeneity. No evidence of affinity maturation was seen upon hyperimmunization. These data favor the conclusion that the restricted IgG response seen in mice hyperimmunized to GAC is not the result of affinity driven competition for antigen among precursor cells. PMID:1092797

1975-01-01

365

Lateral flow immunoassay using magnetoresistive sensors  

NASA Astrophysics Data System (ADS)

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.

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

2009-05-01

366

Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples.  

PubMed

A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples. PMID:10683229

Burestedt, E; Kjellström, S; Emnéus, J; Marko-Varga, G

2000-03-01

367

Enzyme Kinetics  

NSDL National Science Digital Library

This is a lesson from the Computational Biology for Biology Educators workshop series run by the National Computational Science Institute, with sponsorship from the Education Program of the International Supercomputing Conference and the National Science Foundation. The goal of these workshops is the integration of mathematics and computation into the undergraduate Biology classroom, and the integration of biological applications into the Mathematics and Computer Science classroom. Outline Biology background Putting enzymes in action with Agent Sheets Modeling enzymes as dynamic systems with Vensim Biology Background Enzymes accelerate specific molecular events catalytically Movie of MD simulation of HIV-1 protease with substrate There are three types of molecular events that underlie most enzymatic processes Association-dissociation Conformational rearrangement Bond making and breaking Some resources for demonstrating that life\\'s molecular machines are dynamic: Molecules in Motion Database of Macromolecular Movements DSMM - Database of Simulated Molecular Motions The Michaelis-Menten model is a ...

Krause

2008-10-31

368

IgG and IgM antibody reactivity to antigens of Treponema pallidum after treatment of syphilis.  

PubMed

The persistence or loss of IgG and IgM antibody specificities for individual polypeptides of Treponema pallidum after therapy for syphilis was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by the Western blot technique. Both IgG and IgM antibodies to as many as 12 treponemal antigens, including a major 47-kdalton molecule, were evident in plasma from patients with untreated primary syphilis. IgM reactivity declined rapidly and uniformly after therapy, whereas IgG persisted despite some diminution in intensity of staining. Faint-to-moderate IgM and strong IgG antibody reactivities to at least 22 treponemal antigens (12-85 kdaltons) were identified in plasma from patients with untreated secondary and early latent syphilis. Again, IgG antibody declined slightly in staining intensity after treatment but continued to show reactivity against all molecules detected initially. IgM antibody reactivity declined more rapidly and was lost entirely against some determinants, including the 14- and 12-kdalton molecules. Immunofluorescence titers of IgG and IgM antibodies to T. pallidum in sera from these patients generally correlated with results of Western blot analysis. Antibody to the 12-, 14-, and 47-kdalton molecules of T. pallidum may have potential diagnostic applications. PMID:3544253

Baker-Zander, S A; Roddy, R E; Handsfield, H H; Lukehart, S A

1986-01-01

369

Design and Fabrication of a PDMS Microchip Based Immunoassay  

SciTech Connect

In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close a 200µm wide micro channel with flow rate up to 20µl/min.

Shao, Guocheng; Wang, Wanjun; Wang, Jun; Lin, Yuehe

2010-07-01

370

Fluorescence polarization immunoassays for the quantification of caffeine in beverages.  

PubMed

Homogeneous fluorescence polarization immunoassays (FPIAs) were developed and compared for the determination of caffeine in beverages and cosmetics. FPIAs were performed in cuvettes in a spectrometer for kinetic FP measurements as well as in microtiter plates (MTPs) on a multimode reader. Both FPIAs showed measurement ranges in the ?g/L range and were performed within 2 and 20 min, respectively. For the application on real samples, high coefficients of variations (CVs) were observed for the performance in MTPs; the CVs for FPIAs in cuvettes were below 4%. The correlations between this method and reference methods were satisfying. The sensitivity was sufficient for all tested samples including decaffeinated coffee without preconcentration steps. The FPIA in cuvettes allows a fast, precise, and automated quantitative analysis of caffeine in consumer products, whereas FPIAs in MTPs are suitable for semiquantitative high-throughput screenings. Moreover, specific quality criteria for heterogeneous assays were applied to homogeneous immunoassays. PMID:24597592

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

2014-03-19

371

A device architecture for three-dimensional, patterned paper immunoassays.  

PubMed

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care. PMID:25300302

Schonhorn, Jeremy E; Fernandes, Syrena C; Rajaratnam, Anjali; Deraney, Rachel N; Rolland, Jason P; Mace, Charles R

2014-12-21

372

Optical Scanner for Immunoassays With Up-Converting Phosphorescent Labels  

Microsoft Academic Search

A 2-D optical scanner was developed for the imaging and quantification of up-converting phosphor (UCP) labels in immunoassays. With resolution better than 500 mum, a scan rate of 0.4 mm\\/s, and a 1-2% coefficient of variation for repeatability, this scanner achieved a detection limit of fewer than 100 UCP particles in an 8.8 times 104 mum2 area and a dynamic

Janice J. Li; Amy L. Ouellette; Laurent Giovangrandi; David E. Cooper; Antonio J. Ricco; Gregory T. A. Kovacs

2008-01-01

373

Comparison of a noninstrumented immunoassay for carbamazepine to high performance liquid chromatography and fluorescence polarization immunoassay.  

PubMed

The accuracy, precision, and potential clinical utility of a new whole blood, noninstrumented immunochromatographic assay (AccuLevel) for carbamazepine (CBZ) was evaluated in a multicenter trial including 100 pediatric and 205 adult patients. The AccuLevel assay, a fluorescence polarization immunoassay (FPIA), and high-performance liquid chromatography (HPLC) were used to determine CBZ concentration in samples from 111 female and 194 male patients aged 2-72 years (median 25 years). Mean +/- SD plasma CBZ concentrations in all patients were 7.4 +/- 3.0 micrograms/ml with the AccuLevel assay and 7.5 +/- 2.9 micrograms/ml with FPIA. In 204 patients, the mean concentration determined by the HPLC assay was 7.7 +/- 3.0 micrograms/ml, whereas concentrations determined by the AccuLevel and FPIA assays were 8.0 +/- 3.1 and 8.1 +/- 3.1 micrograms/ml, respectively. Concentrations determined by the AccuLevel and FPIA assays were significantly higher than those quantified by HPLC (p less than 0.05), but not different from each other. In addition, the AccuLevel assay was highly correlated with FPIA (r = 0.97) and HPLC (r = 0.98). Coefficients of variation for the AccuLevel assay at 8 micrograms/ml ranged from 6.8 to 7.5% for the three institutions. We conclude that the AccuLevel assay is a simple, reliable method for determining CBZ concentration in a small volume of whole blood and is an acceptable alternative for assessment of CBZ therapy and individualization of CBZ dosage in the physician's office or emergency room. PMID:2369881

Cochran, E B; Massey, K L; Phelps, S J; Cramer, J A; Toftness, B R; Denio, L S; Drake, M E

1990-01-01

374

Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity.  

PubMed

A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion and fenthion. Three haptens with different spacer-arms were synthesized. The haptens were conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens. Rabbits were immunized with the immunogens and six polyclonal antisera were produced and screened against each of the coating antigens using competitive indirect enzyme-linked immunosorbent assay for selecting the proper antiserum. The effect of hapten heterology on immunoassay sensitivity was also studied. The antibody-antigen combination with the most selectivity for malathion was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC(50) values, under optimum conditions, were estimated to be 30.1microgL(-1)for malathion, 28.9microgL(-1) for dimethoate, 88.3microgL(-1) for phenthoate, 159.7microgL(-1) for phosmet, 191.7microgL(-1) for methidathion, 324.0microgL(-1) for fenitrothion, 483.9microgL(-1) for methyl parathion, and 788.9microgL(-1) for fenthion. Recoveries of malathion, dimethoate, phenthoate, phosmet and methidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%. This assay can be used in monitoring studies for the multi-residue determination of O,O-dimethyl organophosphorus pesticides. PMID:18442523

Liang, Ying; Liu, Xian Jin; Liu, Yuan; Yu, Xiang Yang; Fan, Ming Tao

2008-05-19

375

Magneto-controlled electrochemical immunoassay of brevetoxin B in seafood based on guanine-functionalized graphene nanoribbons.  

PubMed

A facile and feasible magneto-controlled immunosensing platform was designed for sensitive electrochemical immunoassay of brevetoxin B (BTX-2) in seafood by using guanine-assembled graphene nanoribbons (GGNRs) as molecular tags on a home-made magnetic carbon paste electrode. Initially, monoclonal mouse anti-BTX-2 antibodies were covalently immobilized on the surface of magnetic beads, which were used as the immunosensing probes for the capture of BTX-2. The recognition elements were prepared by chemical modification of bovine serum albumin-BTX-2 conjugates (BTX-2-BSA) with the GGNRs. Based on a competitive-type immunoassay format, the formed magnetic immunocomplex was integrated on the electrode with an external magnet, followed by determination in pH 6.5 phosphate-buffered solution containing 2 ?M Ru(bpy)(3)Cl(2). Under optimal conditions, the electrochemical signals decreased with the increasing BTX-2 concentrations in the sample. The dynamic concentration range spanned from 1.0 pg mL(-1) to 10 ng mL(-1) with a detection limit of 1.0 pg mL(-1) BTX-2. Inter- and intra-batch assay precisions were substantially improved by resorting to the GGNR manifold. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 95% confidence level were encountered in the analysis of 12 spiked samples including S. constricta, M. senhousia and T. granosa between the electrochemical immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for determination of BTX-2. PMID:22683085

Tang, Juan; Hou, Li; Tang, Dianping; Zhou, Jun; Wang, Zhouping; Li, Jianrong; Chen, Guonan

2012-01-01

376

Food Enzymes  

ERIC Educational Resources Information Center

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…

McBroom, Rachel; Oliver-Hoyo, Maria T.

2007-01-01

377

Engineering enzymes  

PubMed Central

Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of structural and energetic engineering tolerances of the mechanism. Significant barriers to achieving an engineering understanding of enzyme mechanisms arise from natural protein complexity. In certain cases we can surmount these barriers to understanding, such as natural electron tunneling, coupling of electron tunneling to light capture and proton exchange as well as simpler bond breaking redox catalysis. Hope for similar solutions of more complex bioinorganic enzymes is indicated in several papers presented in this Discussion. Armed with an engineering understanding of mechanism, the current serious frustrations to successful creation of functional artificial proteins that are rooted in protein complexity can fall away. Here we discuss the genetic and biological roots of protein complexity and show how to dodge and minimize the effects of complexity. In the best-understood cases, artificial enzymes can be designed from scratch using the simplest of protein scaffolds. PMID:21322497

Moser, Christopher C.

2014-01-01

378

Simple patterned nanofiber scaffolds and its enhanced performance in immunoassay.  

PubMed

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

Wang, Jing; Kang, Qin-shu; Lv, Xiao-guang; Song, Jia; Zhan, Na; Dong, Wei-guo; Huang, Wei-hua

2013-01-01

379

Ultrasensitive immunoassay based on electrochemical measurement of enzymatically produced polyaniline.  

PubMed

A novel ultrasensitive immunoassay method was developed based on the electrochemical measurement of polyaniline, which was catalytically produced by horseradish peroxidase-functionalized gold nanoparticle (HRP-Au NP) probe at an immunosensor. The immunosensor was prepared step-wise by first modifying the electrode with reduced graphene oxide (rGO)/Au NPs nanocomposite followed by the immobilization of capture antibodies on its surface. After performing a sandwich immunoreaction, the quantitatively captured HRP-Au NP nanoprobes could catalyze oxidation of aniline to produce electroactive polyaniline on the immunosensor surface. The electrochemical measurement of polyaniline enabled a novel detection strategy for HRP-based immunoassay. Both the signal amplification of the HRP-Au NP nanoprobe and the electron transfer acceleration of rGO/Au NPs on the immunosensor surface greatly improved the detection sensitivity of the immunoassay method. With the use of human IgG as a model analyte, this method showed a wide linear range over 4 orders of magnitude with a detection limit of 9.7 pg/mL. In addition, the immunosensor had low cost, satisfactory reproducibility and stability, and acceptable reliability. The relatively positive potential range for the polyaniline measurement completely excluded the conventional interference from dissolved oxygen. Thus, this method provides a promising potential for practical applications. PMID:24392763

Lai, Guosong; Zhang, Haili; Tamanna, Tasnuva; Yu, Aimin

2014-02-01

380

Superporous agarose beads as a solid support for microfluidic immunoassay.  

PubMed

We demonstrate here with the feasibility of superporous agarose (SA) beads as a solid support in microfluidic immunoassay by detecting goat IgG. In our procedure, SA beads containing superpores were covalently conjugated to protein A. The conjugated beads were introduced into a polydimethyl siloxane microfluidic device. The sandwich immunoassay was performed in the microfluidic device by subsequently introducing anti-goat IgG as the primary antibodies, goat IgG as analytes, alkaline phosphatase-conjugated F(ab')2 anti-goat IgG as detection antibodies, and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium as substrate in a flow. Depending on the goat IgG concentration, dark and pinky precipitates appeared inside the microchannel immediately after the introduction of all the reagents. The minimum detection limit, 100 pg goat IgG/mL in PBS, was achieved with the naked eye. This enhanced sensitivity is mainly because analytical reagents were allowed to access the outer surface as well as the inner matrices of the beads. This is supported by the facts that the binding of fluorescein isothiocyanate IgG happened throughout the inside matrices of protein A-conjugated SA beads but was limited to the outer surface of protein A-conjugated homogeneous agarose beads. These results suggest that SA beads are highly suitable as a solid support for microfluidic immunoassays. PMID:18550282

Yang, Yoonsun; Nam, Seong-Won; Lee, Nae Yoon; Kim, Youn Sang; Park, Sungsu

2008-09-01

381

Simple Patterned Nanofiber Scaffolds and Its Enhanced Performance in Immunoassay  

PubMed Central

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

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

2013-01-01

382

False-Positive Results Obtained with the Alexon ProSpecT Cryptosporidium Enzyme Immunoassay  

Microsoft Academic Search

Cryptosporidium is known to cause diarrhea in immunocompromised patients and is also associated with outbreaks of disease due to food-borne and waterborne parasites. Traditional procedures, involving iodine staining of wet mounts of stool sediments and trichrome staining, lack the sensitivity to detect Cryptosporidium. Special staining procedures, such as the modified acid-fast and safranin stains, are generally employed. Less labor-intensive antigen

KIRK M. DOING; JILL L. HAMM; JO ANN JELLISON; JESSICA A. MARQUIS; CINDY KINGSBURY

1999-01-01

383

Analytica Chimica Acta 444 (2001) 2736 Development of a class-selective enzyme immunoassay  

E-print Network

in biochemistry, endocrinology or medical chemistry, but also increas- ingly in environmental and toxicological drugs, pesticides or carcinogens [7­10]. The enzy- matically synthesized metabolites are water soluble to an individual toxic compound alone, but rather to a combination of several environmental pollutants. Simultane

Hammock, Bruce D.

384

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 ...

385

Enzyme-Linked Immunoassay for Detection of PCR-Amplified DNA of Legionellae in Bronchoalveolar Fluid  

Microsoft Academic Search

A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5* biotinylated. The amplification product is immobilized on

DANIEL JONAS; ANIKA ROSENBAUM; STEPHAN WEYRICH; ANDSUCHARIT BHAKDI

386

Analytica Chimica Acta 487 (2003) 1529 Optimization and validation of an enzyme immunoassay  

E-print Network

, phenoxyacetic acid herbicides, and photodegradation products of fenoxycarb. Using spiked water samples, assay such as high performance liquid chromatogra- phy (HPLC) [19­24] and gas chromatography (GC) [19. Chemical structure of fenoxycarb (1) and its haptenic derivatives. The haptens contain an amino group

Hammock, Bruce D.

387

Simple fluorescent enzyme immunoassay for detection and quantification of hepatitis C viremia  

Microsoft Academic Search

Background\\/Aims: The viral load of hepatitis C virus, as reflected by hepatitis C virus viremia, has been shown to have important clinical implications. In this study the hepatitis C virus core protein level in serum was evaluated for the detection and quantification of hepatitis C virus viremia.Methods: Hepatitis C virus core protein in serum was detected using a simple and

Takeshi Tanaka; Johnson Y. N. Lau; Masashi Mizokami; Etsuro Orito; Eiji Tanaka; Kendo Kiyosawa; Koichiro Yasui; Yohsuke Ohta; Akira Hasegawa; Satoshi Tanaka; Michinori Kohara

1995-01-01

388

Analytica Chimica Acta 466 (2002) 247256 Development of an enzyme immunoassay for  

E-print Network

- oped. Polyclonal antibodies were generated in rabbits using an isomeric LTXD and iso-LTXD mixture (ELA) or 12-hydroxystearic acid to BSA or ovalbumin (OVA). Various linoleic acid derivatives did not cross react significantly. Using the ovalbumin conjugate of rici- noleic acid as a coating antigen

Hammock, Bruce D.

389

Multicenter Analytical Evaluation of the Automated Electrochemiluminescence Immunoassay for Cyclosporine  

PubMed Central

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

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

2014-01-01

390

HHV-6 A- or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum.  

PubMed

The etiology of multiple sclerosis (MS) remains unknown, but there are indications of a role of human herpesvirus 6 (HHV-6), especially variant A, in the pathogenesis. Higher serum antibody reactivity against an HHV-6 early protein, p41, has been found in MS cases than in controls. The antigen, however, was purified from infected cells with a monoclonal antibody also reactive with a protein (p38) likely to be of cellular origin. To avoid serological crossreactivity with the cellular protein, recombinant p41 proteins from HHV-6A strain GS and HHV-6B strain Z29 were expressed as glutathione-S-transferase fusion proteins (p41-GST), and used as antigens in an enzyme-linked immunosorbent assay (ELISA). p41 variant specific monoclonal antibodies reacted strongly with the respective recombinant proteins. Serum IgM and IgG reactivities with the recombinant p41 antigens were analysed in patients with manifest MS, patients with optic neuritis, patients with other neurological diseases, and in one group of healthy controls. All sera were HHV-6 IgG seropositive by immunofluorescence. The serum IgM or IgG reactivities against the recombinant p41 antigens did not differ significantly between the groups, and the reactivities against the variant A and B antigens were identical. In many samples, the reactivity was very low. The results indicate that p41 is not an optimal target for HHV-6 serology studies, and that the data obtained with the p41 antigen prepared from infected cells (possibly including also p38) should be interpreted with caution. PMID:11793393

Xu, Yunhe; Linde, Annika; Fredrikson, Sten; Dahl, Helena; Winberg, Gösta

2002-03-01

391

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

PubMed

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. PMID:22771742

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

2012-09-01

392

[Gross haematuria as the primary symptom of extramedullary IgM plasmacytoma of the bladder].  

PubMed

Haematuria is the most common clinical symptom of bladder cancer. Besides antibiotic treatment of a probably existing urinary tract infection, ultrasonography of the urinary organs, diagnostic cystoscopy (with biopsy if needed), and radiologic evaluation of the upper urinary tract (intravenous urography, computed tomography or magnetic resonance urography, retrograde pyelography) should be done for further evaluation. Atypical manifestations of systemic diseases with bladder infiltration could feign the clinical appearance of chronic cystitis and hinder determination of the correct diagnosis. The case of a 40-year-old man with recurrent gross haematuria due to extremely rare bladder infiltration through an IgM plasmacytoma is presented. PMID:20383630

Tsaur, I; Probst, M; Chow, K U; Renne, C; Cerovac, I; Pelekanos, C; Juengel, E; Wedel, S A; Bickeböller, R; Karalis, A

2010-07-01

393

Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.  

PubMed

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay. PMID:12437326

Nichkova, Mikaela; Galve, Roger; Marco, M-Pilar

2002-11-01

394

Hapten design and indirect competitive immunoassay for parathion determination: correlation with molecular modeling and principal component analysis.  

PubMed

A novel procedure for parathion hapten design is described. The optimal antigen for parathion was selected after molecular modeling studies of six types of potentially immunizing haptens with the aim to identify the best mimicking target analyte. Heterologous competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed after screening a battery of competitors as coating antigens. The relationship between the heterology degree of the competitor and the resulting immunoassay detectability was investigated according to the electronic similarities of the competitor haptens and the target analyte. Molecular modeling and principal component analysis were performed to understand the electronic distribution and steric parameters of the haptens at their minimum energetic levels. The results suggested that the competitors should have a high heterology to produce assays with good detectability values. An indirect competitive ELISA was finally selected for further investigation. The immunoassay had an IC50 value of 4.79 ng mL(-1) and a limit of detection of 0.31 ng mL(-1). There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%. PMID:17481405

Liu, Yi Hua; Jin, Mao Jun; Gui, Wen Jun; Cheng, Jing Li; Guo, Yi Rong; Zhu, Guo Nian

2007-05-22

395

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

USGS Publications Warehouse

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.

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

2005-01-01

396

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.  

PubMed

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively. PMID:16901856

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

397

Detection of c-reactive protein based on a magnetic immunoassay by using functional magnetic and fluorescent nanoparticles in microplates.  

PubMed

We report the preparation and application of biofunctional nanoparticles to detect C-reactive protein (CRP) in magnetic microplates. A CRP model biomarker was used to test the proposed detection method. Biofunctional magnetic nanoparticles, CRP, and biofunctional fluorescent nanoparticles were used in a sandwich nanoparticle immunoassay. The CRP concentrations in the samples were deduced from the reference plot, using the fluorescence intensity of the sandwich nanoparticle immunoassay. When biofunctional nanoparticles were used to detect CRP, the detection limit was 1.0 ng ml(-1) and the linear range was between 1.18 ng ml(-1) and 11.8 ?g ml(-1). The results revealed that the method involving biofunctional nanoparticles exhibited a lower detection limit and a wider linear range than those of the enzyme-linked immunosorbent assay (ELISA) and most other methods. For CRP measurements of serum samples, the differences between this method and ELISA in CRP measurements of serum samples were less than 13%. The proposed method can reduce the analysis time to one-third that of ELISA. This method demonstrates the potential to replace ELISA for rapidly detecting biomarkers with a low detection limit and a wide dynamic range. PMID:25142023

Yang, S F; Gao, B Z; Tsai, H Y; Fuh, C Bor

2014-11-01

398

Endogenous digoxin-like immunoreactive factors (DLIF) as measured by the CEDIA digoxin assay and a fluorescence polarization immunoassay.  

PubMed

The sensitivity of a new homogeneous enzyme immunoassay for the determination of digoxin (CEDIA Digoxin assay) and a fluorescence polarization immunoassay (FPIA) to interference by digoxin-like immunoreactive factors (DLIF) was studied in sera from pregnant women, newborns, patients undergoing hemodialysis and patients with renal insufficiency, but without hemodialysis. None of the patients had been treated with digoxin or digitoxin. Cross-reactivity of DLIF in the CEDIA assay was generally lower than in the FPIA. Data on the distribution DLIF of values and method comparisons showed that sera of the four patient groups reacted in a completely different way in both assays, suggesting that the nature of DLIF in the four groups is not identical. Addition of digoxin to sera of patients not treated with this drug resulted in a reduction of the apparent DLIF concentration in the CEDIA assay and the FPIA. This shows that DLIF interference may be less pronounced in sera of patients undergoing digoxin therapy compared to untreated persons. Although the CEDIA assay is less sensitive to DLIF interference than the FPIA, further efforts are needed to reduce the extent of this interference. PMID:1509757

Schlebusch, H; Jarausch, J; Domke, I

1992-01-01

399

Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain.  

PubMed

In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species--mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)--and two goose species--domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot. PMID:18568660

Kothlow, Sonja; Haüslaigner, Rafaela; Kaspers, Bernd; Grund, Christian

2008-06-01

400

Fabrication of multiwalled carbon nanotubes-magnetite nanocomposite as an effective ultra-sensing platform for the early screening of nasopharyngeal carcinoma by luminescence immunoassay.  

PubMed

The hybrid nanocomposite that consists of multiwalled carbon nanotubes (MWCNTs) and magnetite (Fe?O4) was fabricated by chemical co-precipitation method. Briefly, CNTs were oxidized with acids to form carboxylic group and then co-precipitated with Fe?O4 to form CNT-Fe?O4 nanocomposites. The nanocomposites were characterized by SEM, HRTEM, XRD, FTIR X-ray photoelectron spectrometry (XPS) and SQUID. The XRD results indicated the high crystallinity of Fe?O? nanoparticles with spinel structure and the transmission electron microscope images depicted the intercalated iron oxide magnetic particles on the surface of CNTs. The MWCNTs-Fe?O? was applied as a sensing interface to perform luminescence enzyme immunoassays. Firstly, EBNA-1 antigen was immobilized onto the carboxyl group functionalized MWCNTs-Fe?O?, followed by binding with anti-EBNA-1 IgA antibodies. The diluted secondary antibodies (anti-human IgA-HRP) were then added to the CNTs/Fe?O?-PEG-EBNA-1-anti-EBV IgA ab complex and act as a catalyst to produce a visible light upon reaction with the substrate luminol. The formed RLU is proportional to the amount of IgA anti-EBV antiobodies on the MWCNTs. The detection limit of proposed CNTs/Fe?O? based luminescence enzyme immunoassay was in the order of 0.00128 EU/mL (1:100,000 fold dilution) for the detection of anti-EBV IgA antibodies, whereas the commercial ELISA and magnetic beads' assay was accounted for up to the dilution fold of 1000 (i.e., 0.128 EU/mL). The initial findings showed that CNTs/Fe?O? nanocomposites have a great potential in luminescent enzyme immunoassays and could be used as a sensing platform for the early screening of nasopharyngeal carcinoma. PMID:24720983

Liu, Chia-Ching; Sadhasivam, S; Savitha, S; Lin, Feng-Huei

2014-05-01

401

The utility of IgG, IgM, and CD138 immunohistochemistry in the evaluation of autoimmune liver diseases.  

PubMed

Primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) present with distinct clinical features. The term "PBC-AIH overlap syndrome (OS)" has been adopted to describe the condition characterized by occurrence of both PBC and AIH, although this clinical entity is difficult to define. This study aimed to assess the utility of IgG, IgM, and CD138 immunohistochemistry in the evaluation of AIH, PBC, and OS. Immunohistochemistry was performed with anti-human IgG, IgM, and CD138 to detect specific plasma cells in the liver. Predominant IgG staining was observed in AIH (85.7 %), while equivocal (46.1 %) or predominant (38.5 %) IgM staining was observed in PBC. In OS, equivocal (20 %) or predominant (80 %) IgG staining was observed. The IgM/IgG ratio was significantly higher in PBC than in AIH or OS (P < 0.005). Histological findings revealed significantly higher IgM expression in PBC at cholangitis activity grades 2-3 compared to those at cholangitis activity grades 0-1. In contrast, a significantly higher IgG expression was observed in PBC at hepatitis activity and fibrosis grades 2-3 compared to those at hepatitis activity and fibrosis grades 0-1. Taken together, periportal plasmacytic infiltrates with variable immunohistochemistry patterns of IgG and IgM expression characterized different autoimmune liver diseases. PMID:24969678

Abe, Kazumichi; Takahashi, Atsushi; Nozawa, Yoshihiro; Imaizumi, Hiromichi; Hayashi, Manabu; Okai, Ken; Kanno, Yukiko; Watanabe, Hiroshi; Ohira, Hiromasa

2014-09-01

402

Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina.  

PubMed

Host defense against opportunistic fungi requires coordination between innate and adaptive immunity for resolution of infection. Antibodies generated in mice vaccinated with the fungus Pneumocystis prevent growth of Pneumocystis organisms within the lungs, but the mechanisms whereby antibodies enhance antifungal host defense are poorly defined. Nearly all species of fungi contain the conserved carbohydrates ?-glucan and chitin within their cell walls, which may be targets of innate and adaptive immunity. In this study, we show that natural IgM antibodies targeting these fungal cell wall carbohydrates are conserved across many species, including fish and mammals. Natural antibodies bind fungal organisms and enhance host defense against Pneumocystis in early stages of infection. IgM antibodies influence recognition of fungal antigen by dendritic cells, increasing their migration to draining pulmonary lymph nodes. IgM antibodies are required for adaptive T helper type 2 (Th2) and Th17 cell differentiation and guide B cell isotype class-switch recombination during host defense against Pneumocystis. These experiments suggest a novel role for the IgM isotype in shaping the earliest steps in recognition and clearance of this fungus. We outline a mechanism whereby serum IgM, containing ancient specificities against conserved fungal antigens, bridges innate and adaptive immunity against fungal organisms. PMID:21149550

Rapaka, Rekha R; Ricks, David M; Alcorn, John F; Chen, Kong; Khader, Shabaana A; Zheng, Mingquan; Plevy, Scott; Bengtén, Eva; Kolls, Jay K

2010-12-20

403

Feedback of kinetic energy into the IGM by supermassive black holes  

NASA Astrophysics Data System (ADS)

Our recent Chandra and XMM-Newton observations of two high redshift broad absorption line (BAL) quasars have revealed the presence of massive outflows of highly ionized, high-metallicity material driven from near the black hole (a few gravitational radii) with velocities of up to 0.4c. The inferred mass outflow rates of 1-10 M? yr-1 and the measured outflow velocities, that significantly exceed the escape velocities of galaxies and clusters of galaxies, imply that quasar winds provide an important feedback mechanism for kinetic energy injection into the IGM. These quasar outflows may also play an important role in the coevolution of black holes and their host galaxies. We present new constraints on the location of the X-ray BAL material based on our analysis of the variability of the ionization properties of the X-ray absorbers. The location and ionization properties of the X-ray BAL material are crucial in inferring the mass outflow rate and rate of kinetic energy injected into the IGM.

Chartas, George; Brandt, W. N.; Gallagher, S. C.

2004-11-01

404

How to Search for Islands of Neutral Hydrogen in the $z \\sim 5.5$ IGM  

E-print Network

Observations of the Lyman-alpha (Ly-$\\alpha$) forest may allow reionization to complete as late as $z \\sim 5.5$, provided the ionization state of the intergalactic medium (IGM) is sufficiently inhomogeneous at these redshifts. In this case, significantly neutral islands may remain amongst highly ionized gas with the ionized regions allowing some transmission through the Ly-$\\alpha$ forest. This possibility has the important virtue that it is eminently testable with existing Ly-$\\alpha$ forest data. In particular, we describe three observable signatures of significantly neutral gas in the $z \\sim 5.5$ 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 ga...

Malloy, Matthew

2014-01-01

405

Bone marrow mastocytosis associated with IgM kappa plasma cell myeloma.  

PubMed

An association between mastocytosis and monoclonal gammopathy is a relatively rare but well recognized clinical finding. In the majority of cases, however, overt myeloma or lymphoma is not detectable morphologically. Here we describe the case of a 51 year-old male patient first presenting with paresis of the right facial nerve and the serological finding of IgM kappa paraproteinemia. The patient did not have organomegaly, lytic bone lesions, or urticaria pigmentosa-type skin lesions. Histological examination of a trephine biopsy specimen revealed the unusual coexistence of plasma cell myeloma and mastocytosis. Immunohistochemically, plasma cells were found to exhibit a monotypic staining for Ig heavy chain mu and Ig light chain kappa, thus confirming their neoplastic nature. Mast cells showed prominent spindling and formed dense multifocal infiltrates, thus enabling the diagnosis of bone marrow mastocytosis. Immunohistochemically, mast cells expressed tryptase, chymase, and KIT (CD117). In addition, aberrant expression of CD25 on mast cells was detected, confirming the coexistence of a neoplastic mast cell-proliferative disorder. According to the WHO proposal for classification of hematopoietic malignancies, this unique case, showing the association of two very rare haematologic neoplasms, can therefore best be referred to as bone marrow mastocytosis associated with IgM kappa plasma cell myeloma (SM-AHNMD). PMID:15160959

Stellmacher, Florian; Sotlar, Karl; Balleisen, Leopold; Valent, Peter; Horny, Hans-Peter

2004-04-01

406

IgM in a Human Neuropathy Related to Paraproteinemia Binds to a Carbohydrate Determinant in the Myelin-Associated Glycoprotein and to a Ganglioside  

Microsoft Academic Search

The IgM in three patients with paraproteinemia and peripheral neuropathy was shown to bind to human myelin-associated glycoprotein (MAG) that had been purified to homogeneity by gel filtration on Sepharose CL-6B. The antigenic determinant reacting with the IgM from all three patients was in the carbohydrate part of the MAG molecule. In addition, the IgM from the same three patients

Amjad A. Ilyas; Richard H. Quarles; Tracy D. Macintosh; Michael J. Dobersen; Bruce D. Trapp; Marinos C. Dalakas; Roscoe O. Brady

1984-01-01

407

The Hitchner B1 strain of Newcastle disease virus induces high levels of IgA, IgG and IgM in newly hatched chicks  

Microsoft Academic Search

Reaseheath line C chicks produced IgA, IgG and IgM in their serum, tears, spleen and Harderian gland (HG) as a consequence of oculotopical vaccination with the Hitchner B1 strain of Newcastle disease virus. The IgM response was seen first, at 5 days after vaccination, and antiviral IgM levels in the tears and serum were negatively correlated to the level of

P. H. Russell; G. O. Ezeifeka

1995-01-01

408

Restriction Enzymes  

NSDL National Science Digital Library

Watson and Crick's description, in 1953, of the double helical structure of the DNA molecule opened the door to a new era in biological understanding and research. Scientists, now knowing the molecular structure of the hereditary molecule, could begin both to elucidate and to manipulate its function. These new studies were, however, dependent on the discovery and use of the many enzymes that can modify or join existing DNA molecules, or aid in the synthesis of new DNA molecules. Includes background article, student activities/demonstratons, graphics, glossary and related references.

BEGIN:VCARD VERSION:2.1 FN:Pamela Peters N:Peters;Pamela ORG:Genentech, Inc. REV:2005-04-14 END:VCARD

1995-06-01

409

Primary enzyme quantitation  

DOEpatents

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.

Saunders, G.C.

1982-03-04

410

Micromotor-based lab-on-chip immunoassays  

NASA Astrophysics Data System (ADS)

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

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