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1

Evaluation of 15 commercial enzyme immunoassays for the detection of rubella-specific IgM  

Microsoft Academic Search

Background: Detection of rubella-specific IgM is a critical investigation in the diagnosis of recent or congenital rubella for which many commercial enzyme immunoassays (EIAs) are now available.Objectives: To evaluate 15 commercially-available EIA kits for the detection of rubella-specific IgM.Study design: A panel of 229 sera was established comprising 72 sera from cases of primary rubella, re-infection, congenital rubella and primary

P. Hudson; P. Morgan-Capner

1996-01-01

2

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

3

Comparative evaluation of three recombinant antigen-based enzyme immunoassays for detection of IgM and IgG antibodies to human parvovirus B19  

Microsoft Academic Search

Background: Diagnosis of acute and past infection with parvovirus B19 is based on detection of IgM and IgG antibodies.Objectives: To evaluate two commercial recombinant antigen-based enzyme immunoassay (EIA) test kits for detection of IgM and IgG antibodies to parvovirus B19 and to compare the commercial EIAs to in-house EIA test procedures.Study design: A panel of 121 sera was used to

Jerry W Pickering; Bagher Forghani; Gordon R Shell; Linxian Wu

1998-01-01

4

Evaluation of Puumala virus IgG and IgM enzyme immunoassays based on recombinant baculovirus-expressed nucleocapsid protein for early nephropathia epidemica diagnosis  

Microsoft Academic Search

Background: Puumala virus (PUU), a member of Hantavirus genus, is the causative agent of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome (HFRS). Rapid diagnosis is essential for clinical management of NE.Objectives: To evaluate the usefulness of recombinant protein-based IgM (direct- and ?-capture) and IgG (direct- and antigen (Ag)-capture) enzyme immunoassays (EIA) in early diagnosis of

Hannimari Kallio-Kokko; Olli Vapalahti; Ĺke Lundkvist; Antti Vaheri

1998-01-01

5

Comparison of four Mycoplasma pneumoniae IgM-, IgG- and IgA-specific enzyme immunoassays in blood donors and patients.  

PubMed

Mycoplasma pneumoniae antibodies were studied in 504 blood donors and 102 patients with infections not caused by M. pneumoniae with the use of enzyme immunoassay kits from ThermoLabsystems (L), Savyon (S), Bio-Rad (B) and Novitec (N). Detection frequencies of M. pneumoniae IgM in blood donors were 14.9% (L), 16.0% (S), 2.8% (B) and 3.8% (N), and in patients were 40.2% (L), 42.2% (S), 9.8% (B) and 16.7% (N). Detection frequencies of M. pneumoniae IgA were 68.5% (L) and 22.8% (S), and in 65 respiratory disease patients were 100% (L) and 53.8% (S). Thus, use of some kits may lead to overdiagnosis of M. pneumoniae infections. PMID:15606638

Csángó, P A; Pedersen, J E; Hess, R D

2004-12-01

6

Evaluation of serological diagnostic indices for mucocutaneous leishmaniasis: immunofluorescence tests and enzyme-linked immunoassays for IgG, IgM and IgA antibodies.  

PubMed Central

The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of immunofluorescence (IF) and enzyme-linked immunoassays (ELISA) for IgG, IgM and IgA antibodies were assessed on sera from mucocutaneous leishmaniasis patients and controls. The sensitivity of the IgG-ELISA test was 93.3% with 95% confidence interval higher than what could be due to a random test not associated with the disease. The specificity of all tests, except the IgM-ELISA, gave indices that could not have been due to chance. The IgG-ELISA and IgG-IF had the highest positive predictive value and the kappa statistic showed that the strength of agreement between the disease and the test was strongest for IgG-ELISA. The IgG-ELISA had a negative predictive value with 95% confidence limits that were not due to chance alone. Efficiency was highest for IgG-ELISA and IgG-IF. These results were obtained using sera from patients with severe or long-standing disease and from controls in whom the disease was ruled out by a negative Montenegro skin test. In field surveys where the differences between cases and controls are less easy to define the diagnostic indices of these tests may vary with the disease prevalence.

Guimaraes, M. C.; Celeste, B. J.; Franco, E. L.; Cuce, L. C.; Belda, W.

1989-01-01

7

Development of Novel Immunoglobulin G (IgG), IgA, and IgM Enzyme Immunoassays Based on Recombinant Puumala and Dobrava Hantavirus Nucleocapsid Proteins?  

PubMed Central

Human infections with Asian and European hantaviruses can result in hemorrhagic fever with renal syndromes of differing severities characterized by renal dysfunction and sometimes by pulmonary symptoms. For the serological detection of human infections by hantaviruses relevant for Europe, we developed monoclonal antibody capture immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays (ELISAs) based on yeast-expressed nucleocapsid proteins of Puumala and Dobrava hantaviruses. Moreover, for diagnosis of acute infections, ?-capture IgM ELISAs were established with nucleocapsid proteins expressed in Drosophila melanogaster Schneider S2 cells. The cutoff values of the ELISAs were determined by investigation of up to 500 human anti-hantavirus-negative serum samples. The specificities of the Puumala and Dobrava virus-specific IgM, IgA, and IgG ELISAs were found to be 100%. The sensitivities of these ELISAs were determined to be 100% with panels of characterized anti-Puumala or anti-Dobrava virus-positive human serum samples. In most cases, Puumala and Dobrava virus infections could be differentiated by ELISA reactivity alone, i.e., endpoint titration with homologous and heterologous antigens.

Meisel, Helga; Wolbert, Anne; Razanskiene, Ausra; Marg, Andreas; Kazaks, Andris; Sasnauskas, Kestutis; Pauli, Georg; Ulrich, Rainer; Kruger, Detlev H.

2006-01-01

8

Homogeneous enzyme immunoassay for netilmicin.  

PubMed Central

A newly developed homogeneous enzyme immunoassay for the determination of netilmicin in serum was evaluated and compared with a radioenzymatic assay. A total of 102 serum samples from patients treated with netilmicin were measured by both methods. This comparison showed an excellent correlation (r = 0.993). The enzyme immunoassay has proved to be precise, accurate, and specific. Because of its rapidity and the ease of performance, this method is a useful alternative to current assays for monitoring serum netilmicin concentrations.

Wenk, M; Hemmann, R; Follath, F

1982-01-01

9

Development of Novel Immunoglobulin G (IgG), IgA, and IgM Enzyme Immunoassays Based on Recombinant Puumala and Dobrava Hantavirus Nucleocapsid Proteins  

Microsoft Academic Search

Human infections with Asian and European hantaviruses can result in hemorrhagic fever with renal syndromes of differing severities characterized by renal dysfunction and sometimes by pulmonary symptoms. For the serological detection of human infections by hantaviruses relevant for Europe, we developed mono- clonal antibody capture immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays (ELISAs) based on yeast-expressed nucleocapsid proteins of

Helga Meisel; Anne Wolbert; Ausra Razanskiene; Andreas Marg; Andris Kazaks; Kestutis Sasnauskas; Georg Pauli; Rainer Ulrich; Detlev H. Kruger

2006-01-01

10

Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays  

PubMed Central

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10?3 to 1.7×10?4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins.

Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Soderlund-Venermo, Maria; Hedman, Klaus

2012-01-01

11

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

12

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

13

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

14

Toxocariasis: serological diagnosis by enzyme immunoassay  

Microsoft Academic Search

An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in

D H de Savigny; A Voller; A W Woodruff

1979-01-01

15

Enzyme immunoassay system for panel testing.  

PubMed

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

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

1989-09-01

16

Heterologous enzyme immunoassay for serum androstenediol  

Microsoft Academic Search

A heterologous enzyme immunoassay for serum androstenediol (Adiol: 3?, 17?-dihydroxy-androst-5-ene) was established. The combination of anti-Adiol antiserum raised in rabbit against Adiol 7-O-(carboxymethyl)oxime (Adiol 7-CMO) conjugated bovine serum albumin (Adiol 7-CMO-BSA) and Adiol 7-iminomethylcarboxylic acid conjugated alkaline phosphatase was used for the assay. The sensitivity of the heterologous assay system was superior to that of a homologous assay system in

Noriko Tagawa; Junko Tamanaka; Aya Fujinami; Toshiko Kiguchi; Takeaki Naito; Toru Takano; Nobuyuki Amino; Yoshiharu Kobayashi

2000-01-01

17

Enzyme Immunoassay Detection of Nitrosomonas europaea  

PubMed Central

An exploratory effort to selectively detect the presence of a nitrifying bacterium, Nitrosomonas europaea, successfully demonstrated the fundamental utility of an enzyme-based immunoassay protocol. The applied polyclonal antibody test seemingly offered a marked improvement over the available analytical options, including plating, activity, and fluorescence immunoassay techniques. Following an initial purification step to enhance overall specificity, this procedure had an apparent lower limit of detection of ?5 × 106 cells per ml. Tests conducted with activated sludge samples exhibited a distinct difference between nitrifying and nonnitrifying mixed liquors, although the highest Nitrosomonas levels observed (i.e., at 1 to 2% of the overall viable cell density) were relatively close to the latter detection boundary.

Saraswat, N.; Alleman, J. E.; Smith, T. J.

1994-01-01

18

Enzyme immunoassay with enhanced specificity for detection of antibodies to Chlamydia trachomatis.  

PubMed Central

Two different methods for preventing the binding of cross-reacting antibodies to the genus-reactive chlamydial lipopolysaccharide (LPS) were used to improve the specificity of an enzyme immunoassay for the determination of antibodies to Chlamydia trachomatis. Coated elementary bodies were treated with either sodium periodate, to oxidize the antigenic sites of the LPS, or Triton X-100, to extract the LPS. By using these new enzyme immunoassays, the standard enzyme immunoassay, and the whole inclusion fluorescence (WIF) assay, antibodies to C. trachomatis were determined in sera from different groups of patients and controls. Paired serum samples from patients with culture-proven urogenital C. trachomatis infections showed similar responses in all three assays. Paired serum samples from patients with Chlamydia psittaci infections showed similar responses in the WIF assay and the standard enzyme immunoassay, whereas significantly reduced titers were obtained in the enzyme immunoassays with treated antigen, especially in the convalescent-phase serum samples. Serum samples from patients with symptoms suggestive of infection with C. trachomatis, pregnant women, and blood donors were evaluated by all three types of assays. Eighty percent of the significant reductions in immunoglobulin G (IgG), IgA, and IgM titers were observed in sera with WIF assay titers in the lower classes (IgG, 1: < or = 256; IgA, 1: < or = 32; IgM, 1: < or = 16). From these results we conclude that oxidation of the antigen by sodium periodate is a simple and effective method of producing an enzyme immunoassay with enhanced specificity that could be useful for diagnostic purposes and seroepidemiological studies.

Ossewaarde, J M; de Vries, A; van den Hoek, J A; van Loon, A M

1994-01-01

19

A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies  

Microsoft Academic Search

New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison with the microimmunofluorescence (MIF) test using specimens from: (i) a group of adult patients

Jacobus M Ossewaarde; Tamara Tuuminen; Wim G Boersma; Mikael Sandström; Pekka Palomäki; Jens Boman

2000-01-01

20

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

PubMed

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

2013-09-25

21

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.

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

2013-01-01

22

[Enzyme immunoassay of usnic acid in lichens].  

PubMed

An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families. PMID:23882952

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

23

Enzyme immunoassays with special reference to ELISA techniques.  

PubMed Central

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

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

1978-01-01

24

Detection of IgM Antibodies to Cytomegalovirus (CMV) Using an Enzyme-labelled Antigen (ELA)  

Microsoft Academic Search

SUMMARY We have applied a peroxidase enzyme-labelled antigen (ELA) for the detection of IgM antibodies to cytomegalovirus: microtitre plates were coated with anti- IgM immunoglobulin. The IgM fraction of human serum was selectively bound to the precoated plates and the virus-specific IgM antibody was then detected by the enzyme-labelled antigen. A very efficient technique for the labelling of virus antigen

H. Schmitz; U. von Deimling; B. Flehmig

1980-01-01

25

Enzyme immunoassay for detection of antibodies against eastern equine encephalomyelitis virus in sentinel chickens.  

PubMed Central

We developed an enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) and IgG subclass antibodies directed against eastern equine encephalomyelitis (EEE) virus in chickens. The assays were compared with the serum plaque reduction neutralization test (PRNT) and the hemagglutination inhibition (HI) test for ability to detect antibodies against EEE virus in laboratory-infected birds. No cross-reactivity was detected in serum from chickens inoculated with St. Louis encephalitis or Highlands J virus. The interval after infection when EEE virus-specific antibodies were first detected by IgM and IgG EIAs was found to be similar to that determined by the PRNT and HI tests: 2 to 4 days. The IgG EIA, PRNT, and HI test detected antibodies to EEE virus for at least 27 to 30 days after inoculation. In contrast, serum from five of seven chickens did not contain detectable IgM 30 days after infection. Similarly, in all three naturally infected sentinel chickens from Maryland, IgM class antibody was undetectable 1 to 5 weeks after IgM was initially detected. EIAs provide simple and rapid alternatives to traditional tests for monitoring EEE virus infections in sentinel chicken flocks. Moreover, the IgM EIA provides a means to separate recently infected chickens from those infected greater than or equal to 1 month earlier.

Olson, J G; Scott, T W; Lorenz, L H; Hubbard, J L

1991-01-01

26

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.

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

27

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

28

Evaluation of the Boehringer Mannheim ES 300 immunoassay analyzer and comparison with enzyme immunoassay, fluorescence polarization immunoassay, and radioimmunoassay methods.  

PubMed

The ES 300 (Boehringer Mannheim Diagnostics, Indianapolis, IN) is a new automated immunoassay analyzer intended for the quantitative determination of a wide range of analytes. We compared its performance to enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), and radioimmunoassay (RIA) methods for cortisol, digoxin, ferritin, prolactin, T4-uptake, total-T3, and TSH. The ES 300 methods showed excellent precision and the manufacturers' linearity claims were met in all cases. Cortisol, prolactin, total-T3, and TSH showed no bias and acceptable correlation with other methods. Digoxin, ferritin and total T4 showed positive bias but acceptable correlation. The ES 300 T4-uptake correlated poorly with the TDx method and showed positive bias; however, these assays appear comparable (although deficient) in diagnostic sensitivity when compared to TSH and T4 data for the same patient population. In all, we found the ES 300 to be an acceptable instrumental alternative for the high volume immunoassay laboratory. PMID:1525980

Camara, P D; Velletri, K; Krupski, M; Rosner, M; Griffiths, W C

1992-08-01

29

Peroxidase-Labelled Monoclonal Antibodies for use in Enzyme Immunoassay  

Microsoft Academic Search

Desirable characteristics of enzyme-antibody conjugates for use in enzyme immunoassay are labelling uniformity, permanent availability and stability. The use of monoclonal antibodies (MCABS) for preparation of enzyme conjugates, in place of poly-clonal antibodies, ensures labelling uniformity and permanent availability. The problem of stability still exists. Monoclonal antibody-horseradish peroxidase (McAB-HREQ) conjugates produced in our laboratory showed variable stability. After extensive testing

D. Henning; K. Nielsen

1987-01-01

30

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

PubMed Central

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

Nielsen, K; Rosenbaum, B; Stiller, J

1985-01-01

31

Development of an Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay for Detection of Equine and Swine IgM Antibodies to Vesicular Stomatitis Virus  

Microsoft Academic Search

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a strepta- vidin-coated ELISA plate. The captured IgM antibodies were detected by application of

EN-MIN ZHOU; JOSE RIVA; ALFONSO CLAVIJO

2001-01-01

32

Rapid detection of Chlamydia trachomatis by an enzyme immunoassay method.  

PubMed

Chlamydia trachomatis has been shown to be a major cause of sexually transmitted diseases in the United States. An enzyme immunoassay (Abbot Laboratories) has been developed that detects chlamydial antigen directly in the urogenital specimens of patients. We have evaluated specimens from 1,074 patients belonging to one of three risk groups. Three swabs were collected from each patient--one each for Neisseria gonorrhoeae, chlamydia cell culture, and enzyme immunoassay. When compared with cell culture, the sensitivity and specificity of the enzyme immunoassay for symptomatic males and females attending a sexually transmitted disease clinic was 82% and 100%, and 91.3% and 95.0%, respectively. A moderate risk group, consisting of female patients seen at either urology or gynecology clinics for genitourinary symptoms was also evaluated. The sensitivity and specificity of the test on this group was 96% and 96.7%. A population of females at low risk were also screened for chlamydial infection. In this group, the sensitivity and specificity of the enzyme immunoassay was 89.3% and 93.2%, respectively. This rapid test is a highly specific and sensitive procedure for the detection of chlamydial antigen in genital specimens from high risk female patients as well as symptomatic males. PMID:3530626

Ryan, R W; Kwasnik, I; Steingrimsson, O; Gudmundsson, J; Thorarinsson, H; Tilton, R C

1986-09-01

33

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...

34

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

PubMed Central

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 TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.

Soldateschi, Dario; Dal Maso, Grazia Maria; Valassina, Marcello; Santini, Laura; Bianchi, Silvia; Cusi, Maria Grazia

1999-01-01

35

Laboratory diagnosis of Toscana virus infection by enzyme immunoassay with recombinant viral nucleoprotein.  

PubMed

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 TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status. PMID:9986827

Soldateschi, D; dal Maso, G M; Valassina, M; Santini, L; Bianchi, S; Cusi, M G

1999-03-01

36

A novel serological technique: polymerase chain reaction enhanced immunoassay. Application to enterovirus IgM diagnosis  

Microsoft Academic Search

The polymerase chain reaction (PCR) method is a sensitive, specific and rapid technique for virus detection. The principles of a PCR enhanced immunoassay (PIA) are described. The method combines solid phase serological techniques with the PCR, providing a versatile and sensitive method for antibody detection. By linking the antigenicity of virus particles with their content of nucleic acid, the method

Robert Aspholm; Shusheng Zuo; Jan Fohlman; Gun Frisk; Göran Friman; Jonas Blomberg

1999-01-01

37

Immunoassay of enzymes--an overview.  

PubMed

Immunochemical techniques with enzymes as the antigen have grown in frequency during the last few years. These techniques have allowed evaluation of enzymes in the presence of endogenous inhibitors. Among those enzymes measured by immunochemical techniques and which have found diagnostic application, mention will be made of alkaline phosphatase (with particular reference to the intestinal, placental, and Regan isoenzymes), lactate dehydrogenase (in which renewed interest has developed due to techniques for specifically measuring the LD-1 isoenzyme), aspartate aminotransferase (of which the cytosolic and mitochondrial forms can now be independently measured by immunochemical techniques), acid phosphatase (for which a specific immunochemical assay for the prostatic enzyme has been widely introduced in diagnostic laboratories), and creatine kinase (for which a variety of immunochemical techniques to measure the M- and B-subunits are now part of standard laboratory assays). Other enzymes which will be discussed in this review include phosphohexose isomerase, amylase, ribonuclease, and lysozyme (muramidase). Finally, the use of enzymes, particularly asparaginase, in the chemotherapy of cancer will be outlined. PMID:6345026

Schwartz, M K

1983-02-01

38

An indirect enzyme immunoassay for the mycotoxin citrinin.  

PubMed Central

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.

Abramson, D; Usleber, E; Martlbauer, E

1995-01-01

39

Dipstick enzyme immunoassay for rinderpest antibody in cattle.  

PubMed

A dipstick enzyme immunoassay (ELISA) has been standardized for the detection of rinderpest antibodies. One hundred and thirty bovine serum samples were analysed by the dipstick ELISA method and the results compared with the conventional plate ELISA method. The sensitivity was found to be similar in both methods. The dipstick ELISA does not require expensive micro-plates and an ELISA reader, and is recommended for use in field laboratories where the qualitative detection of rinderpest antibodies is required. PMID:8273283

Ramadass, P; Meerarani, S; Padmanaban, V D

1993-09-01

40

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

41

Pitfalls and Fallacies of Cat Scratch Disease Serology: Evaluation of Bartonella henselae-Based Indirect Fluorescence Assay and Enzyme-Linked Immunoassay  

Microsoft Academic Search

The diagnostic value of the detection of immunoglobulin G (IgG) and IgM by Bartonella henselae-based indirect fluorescence assay (IFA) and enzyme-linked immunoassay (EIA) for the diagnosis of cat scratch disease (CSD) was evaluated. The IFA was performed either with B. henselae that was cocultivated for a few hours with Vero cells or with noncocultivated B. henselae as the antigen. Additionally,

A. M. C. BERGMANS; M. F. PEETERS; J. F. P. SCHELLEKENS; M. C. VOS; L. J. M. SABBE; J. M. OSSEWAARDE; H. VERBAKEL; H. J. HOOFT; L. M. SCHOULS

42

Enzyme immunoassay validation for the detection of buprenorphine in urine.  

PubMed

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

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

2003-03-01

43

A High Percentage of Serum Samples That Test Reactive by Enzyme Immunoassay for Anti-Brucella Antibodies Are Not Confirmed by the Standard Tube Agglutination Test  

PubMed Central

We describe a retrospective analysis of Brucella enzyme immunoassay (EIA) IgM and IgG results compared to those of the standard tube agglutination test (SAT). Among 1,091 samples tested, 104 (9.5%) and 24 (2.2%) sera were positive by IgM and IgG EIA, respectively. Supplemental testing by SAT showed that 82.7% (86/104) of IgM EIA-reactive samples and 54.2% (13/24) of IgG EIA-reactive samples were negative by SAT. Testing all EIA screen-reactive samples by SAT is required when evaluating patients for potential brucellosis. Due to the limitations of serology, culture remains the gold standard for detecting Brucella infection.

Theel, Elitza S.; Larsen, Staci M.; Patel, Robin

2012-01-01

44

Clinical Specificity of the Enzyme Immunoassay Test for Coccidioidomycosis Varies According to the Reason for Its Performance  

PubMed Central

The diagnosis of coccidioidomycosis relies heavily on serologic test results in addition to clinical history, physical examination, and radiographic findings. Use of the enzyme immunoassay (EIA) has increased because it is rapidly performed and does not require referral to a reference laboratory, as do complement fixation and immunodiffusion tests. However, interpretation of immunoglobulin M (IgM) reactivity by EIA in the absence of immunoglobulin G (IgG) reactivity has been problematic. We conducted a retrospective medical record review of all patients with such IgM reactivity at our institution to identify situations where the finding was more likely to be clinically specific for coccidioidal infection. From 1 January 2004 through 31 December 2008, a total of 1,117 patients had positive EIA coccidioidal serology or EIA IgM-only reactivity; of these, 102 patients (9%) had EIA IgM-only reactivity. Among the 102 patients with EIA IgM-only reactivity, 60 were tested to evaluate symptomatic illness, 13 for follow-up of previously abnormal serology, and 29 for screening purposes. Of the 102 patients, 80 (78%) had positive serologic findings by other methods or had positive culture or histology. Fifty-four (90%) of the 60 patients whose serology was performed to evaluate symptomatic illness had coccidioidal infection, whereas 13 (45%) of 29 patients whose serology was performed for screening purposes had coccidioidal infection. Of the 102 patients with isolated IgM reactivity by EIA, 12 later seroconverted to IgG and IgM reactivity. The use of EIA for screening in 29 asymptomatic persons was associated with unconfirmable results in 13 (45%). Although the majority of patients in our study with isolated IgM reactivity by EIA had probable or confirmed coccidioidomycosis, this result must be interpreted with caution for asymptomatic patients.

Mendoza, Neil; Force, Shannon; Chang, Yu-Hui H.; Grys, Thomas E.

2013-01-01

45

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.

Yoo, Soo Jin; Oh, Hye-Jeon

2007-01-01

46

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

PubMed

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format. PMID:23224576

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

2012-12-07

47

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

48

pH Electrode Based Enzyme Immunoassay for the Determination of Human Chorionic Gonadotropin  

Microsoft Academic Search

The determinazione of HCG is performed by the enzyme immunoassay technique using acetylcholinesterase enzyme as lable and a pH electrode to follow the enzyme activity. The antibody to HCG is immobilized in a membrane form with a polyethylene net; to the sample a certain amount of enzyme-labeled HCG is added, the antibody bound membrane is dipped for two hours; after

Marco Mascini; Fabio Zolesi; Giuseppe Palleschi

1982-01-01

49

Definition of high-proficiency serological markers for diagnosis of varicella-zoster virus infections by enzyme immunoassay.  

PubMed

The levels of Varicella-zoster virus (VZV)-specific IgG, IgM, and IgA antibodies produced in 22 cases of varicella, 22 cases of cutaneous zoster, and 12 cases of acute aseptic meningitis due to VZV in the absence of cutaneous lesions, were measured by indirect enzyme immunoassay (EIA) and compared with those observed in a group of 34 age-matched controls. The definition of cutoff titres for each serological marker and combinations of them allowed early diagnosis of infection in 82% of varicella patients and 91% of patients with acute aseptic meningitis lacking cutaneous lesions on a single serum sample, the specificity being over 90%. The system was as sensitive as the demonstration of intrathecally produced IgG antibodies for the early diagnosis of the infection in 22 cases of neurological disease due to VZV. A working protocol for the serological diagnosis of VZV infections, using currently available EIA reagents, is described. PMID:2542432

Echevarría, J M; de Ory, F; León, P; Téllez, A

1989-03-01

50

Preparation and characterization of digoxin antibody and its application to immunoassays: comparison of performance characteristics between enzyme immunoassay and immunostrip test  

Microsoft Academic Search

After characterizing digoxin immunogens and antibodies, we investigated the assay performances of enzyme immunoassay (EIA), biotin–streptavidin mediated enzyme immunoassay (B-Av EIA), and immunostrip test and compared the effectiveness of these tests for detecting the presence of digoxin and its analogues. The clinical use of digoxin is very complicated due to its narrow therapeutic range. Thus, the development of a rapid

So Young Kim; Myung Ja Choi

2000-01-01

51

Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening.  

PubMed

The newest formulation of the Syva EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine (Roche), utilizing the kinetic interaction of microparticles in solution (KIMS) methodology, RIA tests, and TDx fluorescence polarization immunoassay (FPIA) procedures were compared for marijuana, cocaine, opiates, and barbiturates. Both EMIT II and OnLine immunoassays were performed with a Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, likelihood of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. RIA and OnLine detected 99% of gas chromatography/mass spectrometry (GC/MS)-confirmed marijuana samples; TDx, 95%; and EMIT II, 88%. All four immunoassays detected approximately 99% of confirmed cocaine-positive urines. RIA, OnLine, and TDx all detected 100% of opiate-confirmed samples; EMIT II, 97%. Barbiturate assays exhibited the greatest disparity, with OnLine and TDx detecting 100% of confirmed positives; EMIT II, 88%; and RIA, 78%. For a variety of reasons, we prefer the fully automated EMIT II and OnLine assays for high-volume urine testing, in comparison with our laboratory's semiautomated RIA tests and the limited-throughput TDx system. The four immunoassays investigated delivered comparable performance in terms of detection rates for GC/MS-confirmed positives for some drugs but not for others. PMID:8403399

Armbruster, D A; Schwarzhoff, R H; Hubster, E C; Liserio, M K

1993-10-01

52

Enzyme-Linked Immunoassays for the Detection of Microbial Antigens and Their Antibodies.  

National Technical Information Service (NTIS)

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 unneces...

J. E. Herrmann

1986-01-01

53

Cross-reactivity in the Histoplasma antigen enzyme immunoassay caused by sporotrichosis.  

PubMed

Several endemic mycoses cause cross-reactions in the Histoplasma antigen enzyme immunoassay. Herein, a positive Histoplasma antigen result has been recognized in a patient with sporotrichosis. PMID:21813658

Assi, Maha; Lakkis, Iass E; Wheat, L Joseph

2011-08-03

54

Bioluminescent enzyme immunoassay for the detection of norovirus capsid antigen.  

PubMed

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 10(5) to 10(6) 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

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

2012-10-17

55

Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples  

Microsoft Academic Search

A fast competitive enzyme immunoassay (EIA) for mea- suring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcho- linesterase as enzyme label. Enzyme detection was per- formed by an easy colorimetric assay. Monoclonal anti- bodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 mmol\\/L, a

Frederic Taran; Yveline Frobert; Christophe Creminon; Jacques Grassi; Didier Olichon; Charles Mioskowski; Philippe Pradelles

56

Performance of a Time-Resolved Fluorescence Immunoassay for Measuring Varicella-Zoster Virus Immunoglobulin G Levels in Adults and Comparison with Commercial Enzyme Immunoassays and Merck Glycoprotein Enzyme Immunoassay  

Microsoft Academic Search

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immu- noglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%)

P. A. C. Maple; J. Gray; J. Breuer; G. Kafatos; S. Parker; D. Brown

2006-01-01

57

Coxiella burnetii antibody prevalences among human populations in north-east Africa determined by enzyme immunoassay.  

PubMed

Retrospective serosurveys were conducted to determine the prevalence of antibody to phase-I Coxiella burnetii among humans in various locations of north-east Africa. Sera were tested by the enzyme immunoassay (EIA). Initially the EIA was compared with the standard indirect fluorescent antibody (IFA) method for the detection of antibody to C. burnetii. Results indicated that the EIA was slightly less sensitive (88%), but highly specific (94%) and less subjective than the IFA technique. EIA was subsequently adopted for estimating prevalences in the studied human populations. Data obtained by EIA indicated that the prevalence of C. burnetii antibody among adult Egyptian blood donors was 20% (n = 358) in the Suez Canal area, 16% (n = 501) in the Nile Valley and 10% (n = 427) in the Nile Delta. Among adult patients with acute, undifferentiated fever in Egypt, the prevalence was 28% (n = 50) of acute sera, with seroconversion in 12% of convalescent sera. Antibody to C. burnetii was detected by EIA in the sera of 25% (n = 71) of cattle workers in Egypt, 10% (n = 100) of housewives in Sudan, and 37% (n = 104) of adults in north-west Somalia. Following a fever outbreak affecting all ages in northern Sudan, IgG antibody to C. burnetii was present in 54% of the febrile persons (n = 185) and in 53% of afebrile persons (n = 186). IgM antibody to C. burnetii was demonstrated in 29% of the febrile persons and 15% of the afebrile persons. These results implicate C. burnetii as a possibly important and under-reported cause of human disease and undiagnosed fevers in north-east Africa. PMID:7783275

Botros, B A; Soliman, A K; Salib, A W; Olson, J; Hibbs, R G; Williams, J C; Darwish, M; el Tigani, A; Watts, D M

1995-06-01

58

Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum.  

PubMed

To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2 ng mL(-1). The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations. PMID:22444542

Fu, Xiaoling; Meng, Meng; Zhang, Yu; Yin, Yongmei; Zhang, Xiangsheng; Xi, Rimo

2012-02-13

59

Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay.  

PubMed Central

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.

Landini, M P; Lazzarotto, T; Maine, G T; Ripalti, A; Flanders, R

1995-01-01

60

Comparison of a simplified liquid chromatographic assay of gentamicin in serum with enzyme immunoassay and bioassay  

Microsoft Academic Search

A sensitive and reproducible high-performance liquid chromatographic (HPLC) method is described for assay of gentamicin in serum using ultrafiltration of serum samples and pre-column derivatization with o-phthalaldehyde. The procedure was compared with a bioassay and an enzyme immunoassay. Coefficients of correlation were 0.98 for HPLC vs. bioassay and 0.97 for HPLC vs. immunoassay. The between-day imprecision (n=5) was estimated from

L. Essers

1982-01-01

61

Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis  

PubMed Central

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

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

1999-01-01

62

Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus  

Microsoft Academic Search

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection

K. Nielsen; P. Smith; W. L. Yu; C. Elmgren; P. Nicoletti; B. Perez; R. Bermudez; T. Renteria

2007-01-01

63

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

64

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...

65

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.

Kurstak, Edouard

1985-01-01

66

Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay  

Microsoft Academic Search

Using polyclonal antibodies raised against clathrin, we have developed an enzyme- linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts,

BRUNO GOUD; CHRISTIAN HUET; DANIEL LOUVARD

1985-01-01

67

A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol  

Microsoft Academic Search

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene

Aldo Roda; Anna Chiara Manetta; Francesco Piazza; Patrizia Simoni; Rossella Lelli

2000-01-01

68

Improved sensitivity of an enzyme immunoassay IDEIA for detecting Chlamydia trachomatis  

Microsoft Academic Search

In tests on 375 genital tract specimens a commercially available enzyme immunoassay for Chlamydia trachomatis (IDEIA; Boots-Celltech) was found to have sensitivity values of 62% for men and 74% for women, and a specificity of 97% for both groups, relative to the results obtained by a fluorescence assay (Micro Trak; Syva). The positive predictive value and the negative predictive value

B J Thomas; M F Osborn; C Gilchrist; D Taylor-Robinson

1989-01-01

69

Evaluation of a competitive enzyme immunoassay in screening for syphilis.  

PubMed

A competitive immunoenzymatic method has been developed and evaluated for the serological screening of syphilis. The method detects both anti-Treponema Pallidum IgG and IgM. The kit, commercially known as "Syphilis Screen", is produced by DIESSE Diagnostica Senese (Siena, Italy); all the required reagents are included and are ready for use. The test is performed on undiluted serum and a single incubation step is necessary. The method can be easily automated, and the results do not require a subjective interpretation. A good correlation was found with the Treponema Pallidum Haemagglutination (TPHA) technique: only 14 out of 2350 samples tested (2090 non reactive and 260 reactive) were found to be in disagreement. This test can be considered an alternative to the TPHA method in screening for syphilis. PMID:8673850

De Majo, E; Bianchini, G; Parri, F; Tocci, E; Monaci, M; Paoli, C

1996-01-01

70

Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes.  

PubMed

The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the horseradish peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is presented here. Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an 'enhanced' label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gaining sensitivity (up to 1 order of magnitude) without losing the simplicity of the LFIA format, opening the way to other LFIA applications including their on-demand performance tuning according to the analytical scenario. PMID:22795532

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

2012-06-30

71

Solidphase enzyme immunoassay of urokinase using monoclonal antibodies  

Microsoft Academic Search

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng\\/ml) and evaluation of the enzyme in biological fluids such as urine, pleural elfusions, and ascitic fluids without preliminary

P. Hérion; D. Portetelle; J.-D. Franssen; J. Urbain; A. Bollen

1983-01-01

72

High Seroprevalence of Mycoplasma pneumoniae IgM in Acute Q Fever by Enzyme-Linked Immunosorbent Assay (ELISA)  

PubMed Central

Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

Lai, Chung-Hsu; Chang, Lin-Li; Lin, Jiun-Nong; Chen, Wei-Fang; Kuo, Li-Li; Lin, Hsi-Hsun; Chen, Yen-Hsu

2013-01-01

73

High Seroprevalence of Mycoplasma pneumoniae IgM in Acute Q Fever by Enzyme-Linked Immunosorbent Assay (ELISA).  

PubMed

Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia. PMID:24147043

Lai, Chung-Hsu; Chang, Lin-Li; Lin, Jiun-Nong; Chen, Wei-Fang; Kuo, Li-Li; Lin, Hsi-Hsun; Chen, Yen-Hsu

2013-10-17

74

Enzyme-linked immunosorbent assay (ELISA) for detecting IgM and IgE antibodies in human schistosomiasis.  

PubMed

Sera from patients with acute or early and chronic schistosomiasis were examined for IgG, IgM, and IgE antibody by an enzyme-linked immunosorbent assay technique, using soluble egg antigen from Schistosoma mansoni. Cercarial/adult IgG antibody ratios were determined, using soluble cercarial and adult worm antigens. Sera with cercarial/adult ratios indicative of acute or early schistosomiasis also contained specific IgM antibodies. Schistosome IgE antibody was found in sera from patients with acute schistosomiasis, but in only 1 of 10 sera from patients with chronic schistosomiasis. The inability of ELISA to detect IgE antibodies in chronic sera indicates that it may be a relatively insensitive measure of IgE antibodies in those patients with chronic schistosomiasis. PMID:6986098

Lunde, M N; Ottesen, E A

1980-01-01

75

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

76

[SP-1 in normal and complicated early pregnancy. Diagnosis by enzyme immunoassay].  

PubMed

Pregnancy specific glycoprotein SP-1 was measured by enzyme immunoassay Enzygnost in patients with diagnosis of haemorrhage in early pregnancy. Results were compared with a normal range between 6.-22. week of gestation (n = 268) and were analysed for their predictive value in determining prognosis. 24 of 27 cases with levels below the normal range had an abortion (88,9%) while 28 of 31 women with continuing pregnancies had levels within normal range (90,3%). PMID:6347803

Tatra, G; Bernaschek, G; Tessarek, K H

1983-04-01

77

Semiquantitative Polymerase Chain Reaction Enzyme Immunoassay for the Diagnosis of Pertussis  

Microsoft Academic Search

The two most commonly used targets for diagnosis of pertussis by the polymerase chain reaction have been the pertussis toxin\\u000a promoter and the repeated insertion sequence IS481. A comparative assessment of these primers was performed on routinely collected nasopharyngeal swabs, stored at –20??°C,\\u000a using novel semiquantitative enzyme immunoassays. Both sets of primers behaved similarly with bacterial suspensions, and the\\u000a 17

R. C. Matthews; N. Golbang; W. M. Brück; D. Owen; A. Bailey; V. Weston; J. R. Kerr

1999-01-01

78

An enzyme-immunoassay of abscisic acid in potato ( Solanum commersonii ) cultured cells  

Microsoft Academic Search

Estimation of abscisic acid (ABA) content in potato (Solanum commersonii) suspension-cultured cells with an enzyme-immunoassay (EIA) was investigated. In crude extracts of potato cultured cells or even after simple clean-up using C18 cartridge, EIA based on commercial monoclonal antibodies (Idetek, Inc) failed to detect any ABA content. An interference could be removed by partitioning against ethyl acetate after the C18

Stephen B. Ryu; Paul H. Li; Mark L. Brenner

1992-01-01

79

Detection of Clostridium difficile toxin by enzyme immunoassay, tissue culture test and culture  

Microsoft Academic Search

Summary Clostridium difficile toxin is frequently encountered in patients with antibiotic-associated diarrhea. A commercially available enzyme immunoassay (EIA) detecting toxins A and B was evaluated, screening 148 stool specimens specifically submitted for the detection ofC. difficile. A sensitivity of 100% and a specificity of 97.5% were found compared to a tissue culture assay. The overall prevalence ofC. difficile toxin was

O. Liesenfeld; F. Saeger; H. Hahn

1994-01-01

80

Evaluation of a commercial enzyme immunoassay for detection of norovirus in outbreak specimens  

Microsoft Academic Search

The aim of the study presented here was to use faeces from 41 gastroenteritis outbreaks (130 specimens) in Victoria, Australia,\\u000a to evaluate the sensitivity and specificity of the RIDASCREEN norovirus enzyme immunoassay (EIA) kit relative to reverse transcription-polymerase\\u000a chain reaction and\\/or electron microscopy. Seven specimens known to contain sapovirus, adenovirus, astrovirus and rotavirus\\u000a were also tested. For single-specimen diagnosis the

A. Dimitriadis; J. A. Marshall

2005-01-01

81

European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples  

Microsoft Academic Search

A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription- PCR (RT-PCR). Included in the

Jim J. Gray; Evelyne Kohli; Franco M. Ruggeri; Harry Vennema; Alicia Sanchez-Fauquier; Eckart Schreier; Chris I. Gallimore; Miren Iturriza-Gomara; Helene Giraudon; Pierre Pothier; I. Di Bartolo; N. Inglese; E. de Bruin; B. van der Veer; S. Moreno; V. Montero; M. C. de Llano; M. Hohne; S. M. Diedrich

2007-01-01

82

Evaluation of Enzyme Immunoassay for Detection of Salivary Antibody toHelicobacter pylori  

Microsoft Academic Search

The Helisal test is a quantitative enzyme immunoassay for the measurement of Helicobacter pylori-specific immunoglobulinGantibodiesinsaliva.Thistestwasevaluatedincomparisonwithcultureandhistopathologic examinationofgastricbiopsyspecimensobtainedfrom195patientswhounderwent200endoscopicprocedures for the investigation of gastrointestinal symptoms. Forty-one (21%) patients were found to have peptic ulcer disease, and one other patient had a gastric carcinoma. H. pylori was detected in gastric biopsy specimens obtained from 98 (49%) of the procedures. The sensitivity, specificity, and positive

ANDREW E. SIMOR; EDWARD LIN; FRED SAIBIL; LAWRENCE COHEN; MARIE LOUIE; SUSAN PEAREN; ANDHILDA A. DONHOFFER

1996-01-01

83

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

84

Validation of a simple, sensitive enzyme immunoassay (EIA) for the determination of caprine plasma LH  

Microsoft Academic Search

The study describes the development and validation of a simple and highly sensitive enzyme immunoassay (EIA) for the determination of goat plasma LH, utilizing the biotin–streptavidin peroxidase amplification system in a competitive-binding assay. Micro-titer plates were coated with goat anti-rabbit globulin as the second antibody and biotin was coupled to oLH and used as a bridge between streptavidin peroxidase and

A. Haldar; R. Paul; S. Pan; A. Mitra; C. Biswas; D. Majumdar; S. Ghosh; N. P. Singh; S. V. Ngachan; K. M. Bajurbhoruea; B. S. Prakash

2009-01-01

85

A Competitive Enzyme Immunoassay for Albuterol: Its Application for the Drug Screening in Urine  

Microsoft Academic Search

A competitive enzyme immunoassay using purified monoclonal IgGl and an alkaline phosphatase-albuterol derivative has been developed for the quantification of albuterol in urine. The calibration curve obtained in optimal incubation, conditions is characterized by a minimum detectable level of 26 fmol\\/well and a working range from 52 fmol to 4,2 pmol\\/well. This method allows the precise and accurate quantification of

Albert Adam; Huy Ong; Andre Gravel; Jacques Messier; Monique Bellemare; Francine Lantin; Gilles Sauvé; Peeter Tyssen

1991-01-01

86

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

87

Application of magnetic nanoparticles in full-automated chemiluminescent enzyme immunoassay  

Microsoft Academic Search

The magnetic nanoparticles (MNPs) Therma-Max™ were used as a carrier to develop an automated sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone (TSH) in a sensitive and specific way. The Therma-Max™ particles allow for automation because, unlike magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate automatic handling. Signal intensities detected with MNPs were 8-fold

Xiaomao Xie; Noriyuki Ohnishi; Yuki Takahashi; Akihiko Kondo

2009-01-01

88

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

89

Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique.  

PubMed

Two types of flexible, sensitive and rapid competitive flow injection enzyme immunoassay were developed and evaluated for their potential use in the bioanalysis of steroids. Instead of the more typical approach where the signal is generated from antibody-bound hapten-enzyme conjugate, the non-bound fraction passing through the affinity column was allowed to react with an enzyme substrate from a merging channel in a post-column reaction system. The enzyme product (p-aminophenol or 4-methyl umbelliferol) was amperometrically or fluorometrically detected. Several parameters known to affect signal generation in the immunoassay were evaluated, including flow rate through the affinity column and through the reaction coil, the length of the reaction coil and of the affinity column. In the pre-incubation approach, where samples were mixed with enzyme conjugate and antibodies before injection, a sample throughput as high as 20 h(-1) was possible. The signal precision was about 1% (RSD) for cortisol (0.6-80 pmol) and 2% (RSD) for budesonide (0.02-12.5 pmol). In the displacement assay for cortisol, enzyme-labelled analyte was displaced from the affinity column when the sample was injected into the flow. A standard curve was obtained with a signal precision of 4-20% for 12.5-1250 pmol injected. The same instrumental set-up was used in both types of immunoassay, and thus a highly flexible system was obtained. A simple replacement of the affinity column from protein G in the pre-incubation approach to a column containing primary antibodies in the displacement assay was needed. PMID:9005953

Kronkvist, K; Lövgren, U; Svenson, J; Edholm, L E; Johansson, G

1997-01-15

90

Detection of Clostridium difficile toxins by enzyme immunoassay.  

PubMed Central

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.

Krishnan, C.

1986-01-01

91

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

PubMed

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an antibiotic forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20-IC80) equaled 0.03-0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05 ng/ml, 2.9 ng/ml, and 0.26-32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%. PMID:21442924

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

92

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

NASA Astrophysics Data System (ADS)

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

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

93

Quantitation of human serum polymeric IgA, IgA1 and IgA2 immunoglobulin by enzyme immunoassay.  

PubMed Central

This report concerns the relative quantitation of serum polymeric IgA and polymeric IgA subclass concentrations by enzyme immunoassay (EIA). The assay relies on the specific binding of polymeric IgA to secretory component. Competition between pentameric IgM and polymeric IgA for binding to secretory component was observed. Thus, samples were adsorbed for IgM by affinity chromatography before the EIA was performed. The assay was used to determine an age-related range of serum polymeric IgA concentrations and to compare the polymeric IgA concentrations in patients with IgA nephropathy (n = 50) to those of controls (n = 50). The serum concentrations of both polymeric IgA and polymeric IgA1 increased with age reaching adult values of around 12 years of age. Polymeric IgA2 concentrations did not reach adult levels until 18 years of age. The ratio of the polymeric IgA concentration to the total serum IgA concentration was found to be significantly increased in children under 2 years of age compared with those over 4 years of age (Mann-Whitney U-test, P less than 0.01). Patients with IgA nephropathy had significantly increased concentrations of polymeric IgA (P = 0.001) and polymeric IgA1 (P = 0.001) but similar polymeric IgA2 concentrations to controls.

Jones, C; Mermelstein, N; Kincaid-Smith, P; Powell, H; Roberton, D

1988-01-01

94

Evaluation of Four Commercial Immunoglobulin G (IgG)- and IgM-Specific Enzyme Immunoassays for Diagnosis of Mycoplasma pneumoniae Infections  

PubMed Central

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting Mycoplasma pneumoniae immunoglobulin G (IgG)- and IgM-specific antibodies Platelia EIA, ImmunoWELL M. pneumoniae ELISA IgG and IgM, ETI-MP-IgG and IgM EIAs and Biotest anti-M. pneumoniae IgG and IgM ELISA (referred to herein as EIA-Platelia, EIA-BMD, EIA-Sorin, and EIA-Biotest). Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections who tested positive by PCR for M. pneumoniae in respiratory specimens (group I; 52 serum samples), 61 healthy children and adults (group II; 61 serum samples), and 20 patients with rheumatoid factor or antinuclear antibodies, or who tested positive for antiviral IgM (group III; 20 serum samples). In group III, the IgM specificity for EIA-Platelia, EIA-BMD, EIA-Biotest, and EIA-Sorin was 100, 90, 65, and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivities (89 to 92%); the sensitivity for IgG was greater with EIA-BMD and EIA-Biotest than with EIA-Platelia and EIA-Sorin (66 and 78% versus 55 and 52%, respectively). In adult patients from group I, 9 to 10 serum samples were positive for IgG with a concordant sensitivity of 75 to 83% between the four EIAs but a striking difference in IgM sensitivity: 16% by EIA-Platelia and EIA-BMD, 50% by EIA-Biotest, and 58% by EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIAs and making them inaccurate for routine diagnosis. A good concordance of IgG seroprevalence in healthy adults was found between the four EIAs (66 to 70%), though this concordance was lower with EIA-Platelia (43%). In healthy children, EIA-BMD and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former compared to 17 and 20%, respectively, for the latter). These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of M. pneumoniae infections in children, as long as the EIA used is specific. In adults, the difficult interpretation of EIAs suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.

Petitjean, J.; Vabret, A.; Gouarin, S.; Freymuth, F.

2002-01-01

95

Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin-luciferase bioluminescence.  

PubMed

Firefly luciferin-luciferase bioluminescence is known for its high quantum yield (41.0 ± 7.4%). Given this high quantum yield, application of this bioluminescence is expected to be useful in the field of clinical diagnostics. The kinetic profile of this bioluminescence exhibits an instant rise (<1 s) and a rapid decay in light emission (decreased to 42% after 5 s). In this study, we applied four enhancers including coenzyme A, inosine5'-triphosphate sodium salt, sodium tripolyphosphate and potassium pyrophosphate to prolong light emission. When these enhancers were used, luminescence was only decreased to 89, 83, 87 and 82% after 5 s, respectively. These materials modified the kinetic profile of bioluminescence so that the luminescence is more suitable for clinical application. It becomes more suitable because they enable highly sensitive integration and simplification of a device by separating luminescence measurements from dispensing of reagents. Using these enhancers, we then developed a bioluminescent enzyme immunoassay (BLEIA) for hepatitis B virus surface antigen (HBsAg) that employed firefly luciferase as a labeling enzyme. We compared the results obtained from the HBsAg BLEIA method with the conventional chemiluminescent enzyme immunoassay method, and found a satisfactory correlation (r=0.984, n=118). PMID:21681909

Minekawa, Takayuki; Ohkuma, Hiroshi; Abe, Katsushi; Maekawa, Hiroaki; Arakawa, Hidetoshi

2010-03-30

96

Cross-Reactivity of the PLATELIA CANDIDA Antigen Detection Enzyme Immunoassay with Fungal Antigen Extracts  

PubMed Central

We studied the specificity of the PLATELIA CANDIDA Ag enzyme immunoassay by using 130 isolates of 63 clinically relevant fungal species. Antigen extracts of seven Candida spp. (Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis) repeatedly yielded positive reactions (>0.5 ng/ml). Geotrichum candidum and Fusarium verticillioides were found to yield borderline-positive reactions (0.25 to 0.50 ng/ml). Antigen preparations from the other 54 fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi, did not cross-react in the assay.

Rimek, Dagmar; Singh, Jagpal; Kappe, Reinhard

2003-01-01

97

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

98

Interlaboratory comparison of Cry1Ab toxin quantification in MON 810 maize by enzyme-immunoassay  

Microsoft Academic Search

A laboratory ring trial was performed in four laboratories for determination of Cry1Ab toxin in leaf material of MON 810 maize using a standardised enzyme-linked immunoassay protocol. Statistical analysis was carried out by the ISO 5725-2 guidelines, sample standard deviation and standard error, within-laboratory and inter-laboratory SD and SE were calculated. Measured inter-laboratory average values were 12.5±4.0, 15.3±4.6 and 72.6±17.8

András Székács; Gabriele Weiss; David Quist; Eszter Takács; Béla Darvas; Matthias Meier; Trilochan Swain; Angelika Hilbeck

2012-01-01

99

Interlaboratory comparison of Cry1Ab toxin quantification in MON 810 maize by enzyme-immunoassay  

Microsoft Academic Search

A laboratory ring trial was performed in four laboratories for determination of Cry1Ab toxin in leaf material of MON 810 maize using a standardised enzyme-linked immunoassay protocol. Statistical analysis was carried out by the ISO 5725-2 guidelines, sample standard deviation and standard error, within-laboratory and inter-laboratory SD and SE were calculated. Measured inter-laboratory average values were 12.5±4.0, 15.3±4.6 and 72.6±17.8

András Székács; Gabriele Weiss; David Quist; Eszter Takács; Béla Darvas; Matthias Meier; Trilochan Swain; Angelika Hilbeck

2011-01-01

100

Application of magnetic nanoparticles in full-automated chemiluminescent enzyme immunoassay  

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

101

Determination of tumor necrosis factor-? (TNF-?) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay  

Microsoft Academic Search

Objectives: To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-? (TNF-?).Methods: The method is based on the use of two monoclonal antibodies against TNF-?, one “capture” antibody and one labeled with biotin, in a “sandwich type” assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves

Constantinos Petrovas; Spiros M Daskas; Evriklia S Lianidou

1999-01-01

102

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals  

Microsoft Academic Search

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This

K. Nielsen; P. Smith; W. L. Yu; C. Elmgren; G. Halbert; P. Nicoletti; B. Perez; S. Conde; L. Samartino; A. Nicola; R. Bermudez; T. Renteria

2008-01-01

103

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

PubMed Central

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

De Saeger, S; Van Peteghem, C

1996-01-01

104

Performance of a Time-Resolved Fluorescence Immunoassay for Measuring Varicella-Zoster Virus Immunoglobulin G Levels in Adults and Comparison with Commercial Enzyme Immunoassays and Merck Glycoprotein Enzyme Immunoassay  

PubMed Central

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

Maple, P. A. C.; Gray, J.; Breuer, J.; Kafatos, G.; Parker, S.; Brown, D.

2006-01-01

105

Human rotavirus antigen detection by enzyme-immunoassay with antisera against Nebraska calf diarrhoea virus.  

PubMed Central

A four-layer solid phase enzyme-immunoassay (EIA) with antisera against Nebraska calf diarrhoea virus (NCDV) as immunoreagents was developed to detect human rotavirus antigens from stool specimens of patients with acute rotavirus gastroenteritis. Polystyrene beads were used as the solid phase, guinea-pig and rabbit anti-NCDV immunoglobulin as the catching and secondary antibody, and peroxidase-conjugated swine anti-rabbit immunoglobulin as the indicator antibody. A comparison of the developed NCDV-EIA with an identical EIA, using antisera against human rotavirus (HRV-EIA) instead of NCDV antisera, was made with 216 stool specimens positive or negative for rotavirus. A complete agreement was obtained between the two methods provided that appropriate confirmatory tests were included. The developed NCDV-EIA was as sensitive and specific for rotavirus as the HRV-EIA, and it allowed the detection of both established rotavirus types 1 and 2 from stools with equal sensitivity. The difficulties in cultivating human rotavirus in vitro for immunisation and the relative ease of growing NCDV in widely-used continuous cell lines make NCDV a good alternative in the preparation of the highly specific and sensitive rotavirus antisera required in immunoassays, and facilitate the setting-up methods for the routine diagnosis of rotavirus gastroenteritis by EIA or RIA in diagnostic virus laboratories.

Sarkkinen, H K

1981-01-01

106

Evaluation of an Enzyme Immunoassay for Detection of the Clostridium difficile Common Antigen, Glutamate Dehydrogenase (GDH), in Fecal Specimens  

Microsoft Academic Search

C. difficile is reported to cause up to 25% of cases of antibiotic-associated diarrhea (AAD) and most cases of pseudomembranous colitis (PMC). Laboratory diagnosis typically consists of toxin detection in fecal specimens by enzyme immunoassay (EIA) or tissue culture cytotoxin assay (CTA). Culture on C. difficile selective media offers the highest sensitivity, but has low specificity due to detection of

M. D. Poulter; M. Z. Doymaz; L. Zheng; C. Flores; V. Caruthers; P. LeBourgeois; L. Gibson; D. A. Bruckner

107

Enzyme Immunoaffinity Chromatography—A Rapid Semi-quantitative Immunoassay Technique for Screening the Presence of Isoproturon in Water Samples  

Microsoft Academic Search

Immunochromatography devices based on the principles of affinity chromatography and enzyme immunoassay have been developed to illustrate the possibility of providing extra-laboratory 'in the field' tests for pesticide monitoring. Isoproturon was chosen in this study as an example though other pesticides could have been used provided a suitable antiserum existed. The test system was prepared by immobilising isoproturon antibodies to

M. Fawaz Katmeh; A. J. Miguel Godfrey; Derek Stevenson; G. Wynne Aherne

1997-01-01

108

Performance Characteristics of the Platelia Aspergillus Enzyme Immunoassay for Detection of Aspergillus Galactomannan Antigen in Bronchoalveolar Lavage Fluid  

Microsoft Academic Search

We have evaluated the Platelia Aspergillus enzyme immunoassay for detection of galactomannan in bron- choalveolar lavage (BAL) specimens in solid organ transplant patients with aspergillosis. The precision and reproducibility in serum or BAL to which galactomannan was added were similar. Sensitivity was 81.8% in patients with aspergillosis, and specificity was 95.8% in lung transplant patients who underwent BAL for surveillance

S. Husain; C. J. Clancy; M. H. Nguyen; S. Swartzentruber; H. Leather; A. M. LeMonte; M. M. Durkin; K. S. Knox; C. A. Hage; C. Bentsen; N. Singh; J. R. Wingard; L. J. Wheat

2008-01-01

109

Development and Evaluation of a Chromatographic Procedure for Partial Purification of Substance P with Quantitation by an Enzyme Immunoassay  

Microsoft Academic Search

We have developed a simple chromatographic procedure for the partial purification of substance P (SP) from acidified plasma and serum samples. We have evaluated a sensitive antigen competition enzyme immunoassay (EIA) for the quantitation of SP. The chromatographic procedure has recovery efficiencies ranging from 94.8 to 125%. The immunoreactivity of unknown amounts of purified SP subjected to the preparative procedure

WILLIAM P. FEHDER; WEN-ZHE HO; DONALD E. CAMPBELL; WALLACE W. TOURTELLOTTE; LISA MICHAELS; JOANN R. CUTILLI; MARINA UVAYDOVA; STEVEN D. DOUGLAS; Joseph Stokes Jr

1998-01-01

110

Comparison of chemiluminescence enzyme immunoassay (CLEIA) with ELISA for the determination of anti-cyclic citrullinated peptide antibodies  

Microsoft Academic Search

BackgroundAutoantibodies against cyclic citrullinated peptide (anti-CCP) are sensitive and highly specific markers for rheumatoid arthritis (RA). We evaluated the analytical and diagnostic accuracy of chemiluminescence enzyme immunoassay (CLEIA) for anti-CCP antibodies, and compared it with that of ELISA.

Ryo Tanaka; Masao Takemura; Masao Sato; Yasunori Yamada; Takahito Nakagawa; Takuro Horibe; Masato Hoshi; Hirofumi Otaki; Hiroyasu Ito; Mitsuru Seishima; Katsuji Shimizu

2010-01-01

111

Anti-cyclic citrullinated peptide autoantibodies measured by an automated enzyme immunoassay: Analytical performance and clinical correlations  

Microsoft Academic Search

BackgroundAutoantibodies against cyclic citrullinated peptide (anti-CCP) are considered to be a sensitive and specific marker for rheumatoid arthritis (RA). This study evaluated the analytical performance and clinical correlation of an automated enzyme immunoassay (DSX, DINEX Technologies), for the detection of anti-CCP autoantibodies (DIASTAT™ anti-CCP, Axis-Shield, DUNDEE UK).

Marilina Tampoia; Vincenzo Brescia; Antonietta Fontana; Piera Maggiolini; Giovanni Lapadula; Nicola Pansini

2005-01-01

112

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

113

New enzyme immunoassays for the serologic detection of woodchuck hepatitis virus infection.  

PubMed

The woodchuck and the woodchuck hepatitis virus (WHV) have been used as a model of hepatitis B virus infection and its disease sequelas. Serologic responses to WHV infection have been described in previous reports from this laboratory by using virus-specific radioimmunoassays (RIAs) for WHV surface antigen, antibody to WHV core antigen, and antibody to WHsAg. In this study, we developed and evaluated new enzyme immunoassays (EIAs) for these WHV serologic markers. Relative to the established RIAs, the EIAs were either improved or comparable in their sensitivity and specificity, and in their utility for monitoring experimental WHV infection and classifying woodchucks into serological diagnostic categories. These EIA systems are amenable to the quantitative titration of antibodies and quantitation of WHV antigens in serum, and ultimately should allow improved resolution of virologic and humoral immune responses of woodchucks to WHV infection. PMID:8216715

Cote, P J; Roneker, C; Cass, K; Schödel, F; Peterson, D; Tennant, B; De Noronha, F; Gerin, J

1993-01-01

114

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. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23785024

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

2013-06-20

115

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

PubMed

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. PMID:11234804

Thurman, E M; Aga, D S

116

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

PubMed Central

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

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

1992-01-01

117

Detection and quantitation of chloramphenicol by competitive enzyme-linked immunoassay.  

PubMed Central

A competitive enzyme-linked immunoassay for the detection and quantitation of chloramphenicol has been developed. The binding of specific rabbit antibody to solid-phase-bound chloramphenicol was competitively inhibited by free chloramphenicol in the sample to be assayed. Antibody not displaced was indicated by using a commercially available, enzyme-linked, anti-rabbit immunoglobulin preparation and reacted with added substrate. Enzyme activity, measured spectrophotometrically, was inversely proportional to the concentration of chloramphenicol in the sample. Quantitation of the antibiotic was linear to 100 ng/ml, with a lower limit of detection of 1 ng/ml (P less than 0.05). Specificity was demonstrated by the lack of inhibition by any of 31 selected antimicrobial agents or chemicals tested in the assay. Chloramphenicol sodium succinate and thiamphenicol, an experimental antibiotic similar in structure to chloramphenicol, were the only drugs found to produce cross-reactions. In addition to excellent sensitivity and specificity, the assay was shown to have good precision and economy and could be completed in approximately 24h.

Campbell, G S; Mageau, R P; Schwab, B; Johnston, R W

1984-01-01

118

Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus.  

PubMed

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA. PMID:17467200

Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Nicoletti, P; Perez, B; Bermudez, R; Renteria, T

2007-03-30

119

Staphylococcal IgM enzyme-linked immunosorbent assay for diagnosis of periprosthetic joint infections.  

PubMed

Delayed orthopedic joint prosthesis infections (DOJP-Is) due to staphylococci frequently result in prosthetic revision. Specific and noninvasive diagnostic tests are unavailable, and DOJP-Is are commonly diagnosed at advanced stages of disease. An enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies against staphylococcal slime polysaccharide antigens. Using a cutoff of 0.35 ELISA units, the test showed a specificity of 95.1% (95% confidence interval [CI], 85.4 to 98.7%) and a sensitivity of 89.7% (71.5 to 97.3%) on a sample of 90 individuals. PMID:21068292

Artini, Marco; Romanň, Carlo; Manzoli, Lamberto; Scoarughi, Gian Luca; Papa, Rosanna; Meani, Enzo; Drago, Lorenzo; Selan, Laura

2010-11-10

120

Staphylococcal IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Periprosthetic Joint Infections ? ?  

PubMed Central

Delayed orthopedic joint prosthesis infections (DOJP-Is) due to staphylococci frequently result in prosthetic revision. Specific and noninvasive diagnostic tests are unavailable, and DOJP-Is are commonly diagnosed at advanced stages of disease. An enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies against staphylococcal slime polysaccharide antigens. Using a cutoff of 0.35 ELISA units, the test showed a specificity of 95.1% (95% confidence interval [CI], 85.4 to 98.7%) and a sensitivity of 89.7% (71.5 to 97.3%) on a sample of 90 individuals.

Artini, Marco; Romano, Carlo; Manzoli, Lamberto; Scoarughi, Gian Luca; Papa, Rosanna; Meani, Enzo; Drago, Lorenzo; Selan, Laura

2011-01-01

121

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

NASA Astrophysics Data System (ADS)

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

Hock, Bertold; Giersch, Thomas; Kramer, Karl

1993-03-01

122

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

2012-08-09

123

Detection of culture-derived Babesia bovis exoantigen using a two-site enzyme immunoassay.  

PubMed Central

Soluble exoantigens in the supernatants of Babesia bovis cultures have been shown to be efficient immunogens against bovine babesiosis. We used a two-site enzyme immunoassay to monitor the release of these antigens during in vitro cultivation. Bovine immunoglobulin G was isolated from serum of an adult cow previously immunized with culture-derived B. bovis exoantigens and challenged via needle with virulent parasites. The specific immunoglobulin G was used as a capture antibody and as an enzyme-conjugated recognizing antibody. The optimal protein concentration of capture antibody was 10 micrograms/ml. The 24-h cultures showed the greatest antigen concentration. The test was sensitive for detection of differences in species-specific antigenic activity among B. bovis isolates, for determining loss of antigenicity during storage and formalinization, and for monitoring the kinetics of exoantigen release during in vitro cultivation. Antigens cross-reactive with the other major Babesia species of cattle, Babesia bigemina, were also detected with this assay. The high specificity, sensitivity, and reproducibility of this technique should facilitate detection and quantitation of Babesia antigens during purification and in standardization of candidate immunogens.

Montealegre, F; Montenegro-James, S; Kakoma, I; Ristic, M

1987-01-01

124

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

PubMed Central

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

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

2006-01-01

125

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

PubMed

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

Märtlbauer, E; Gareis, M; Terplan, G

1988-01-01

126

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

PubMed

The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses. PMID:24148422

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

2013-06-15

127

Dot-enzyme immunoassay for visual detection of antibodies to pseudorabies virus in swine serum.  

PubMed Central

A modified solid-phase enzyme immunoassay (EIA) is described for the visual detection of anti-pseudorabies virus (anti-PRV) antibody in porcine serum. Dots of PRV antigens were adsorbed to nitrocellulose paper (hence the name dot-EIA), and the remaining nonspecifically reactive sites were blocked with bovine serum albumin or skim milk powder. After immersion in test serum, bound antibodies were reacted with a peroxidase-conjugated anti-porcine immunoglobulin G (H & L). Positive reactions were easily visualized as brown dots after enzyme degradation of a substrate containing hydrogen peroxide and diaminobenzidine. The dot-EIA was comparable to the serum neutralization test and the standard microtiter EIA in its ability to detect antibody in the sera of pigs 9 days after experimental infection and 12 days after contact with infected pigs. The sensitivity and specificity of the dot-EIA relative to the serum neutralization test and the standard EIA were determined from the testing of 856 field sera from the United Kingdom, the United States, and Canada. In all comparisons, both the relative sensitivity and specificity of the dot-EIA were in the order of 98 to 99%. The dot EIA appears to have potential application as a rapid and economical field test in the diagnosis of PRV infection. Images

Afshar, A; Wright, P F; Dulac, G C

1986-01-01

128

Effect of Multiple Freeze and Thaw Cycles on the Sensitivity of IgG and IgM Immunoglobulins in the Sera of Patients With Syphilis.  

PubMed

We describe the effects of multiple freeze and thaw cycles on the sensitivity of the immunoglobulins IgG and IgM measured by enzyme-linked immunoassays in the sera of patients with syphilis. Stored frozen sera can withstand repeated freezing and thawing cycles with a minimal detrimental effect on the sensitivity of the sera. PMID:24113410

Castro, Arnold R; Jost, Heather A

2013-11-01

129

Production and Evaluation of Reagents for Detection of Histoplasma capsulatum Antigenuria by Enzyme Immunoassay?  

PubMed Central

The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the A450 with antigen divided by the A450 without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units ± standard deviation, 14.9 ± 0.6 versus 6.4 ± 0.4 for rabbits immunized with C-Ag versus 2.4 ± 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen (P < 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test (P < 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.

Lindsley, Mark D.; Holland, Heather L.; Bragg, Sandra L.; Hurst, Steven F.; Wannemuehler, Kathleen A.; Morrison, Christine J.

2007-01-01

130

An enzyme immunoassay for immunoglobulin M antibodies to Toxoplasma gondii which is not affected by rheumatoid factor or immunoglobulin G antibodies.  

PubMed Central

An enzyme-linked immunosorbent assay (ELISA) for total antibodies to Toxoplasma gondii was modified to measure specific immunoglobulin M (IgM) antibodies. The assay requires three incubation periods totaling 2 h and enzyme-labeled-heavy-chain-specific antibodies to human IgM. The objective read-out in absorbance was normalized to percent of a standardized positive control for interpretations. No difference was observed between the assay results with or without previous absorption of the samples by Staphylococcus aureus protein A to remove most of the IgG antibodies. Addition of serum containing very high levels of IgG antibodies to another containing both IgG and IgM antibodies did not change the IgM assay values for the latter. None of the 22 sera containing high levels of IgM rheumatoid factor (RF) gave positive ELISA IgM results, even though 8 of them also had high levels of IgG toxoplasma antibodies. Mixtures of sera containing high concentrations of RF with sera having high levels of IgG toxoplasma antibodies also failed to show any false-positive reactions in the IgM toxoplasma assay. Thus, this ELISA for T. gondii IgM antibodies was not affected by IgG toxoplasma antibodies and RF.

Lin, T M; Chin-See, M W; Halbert, S P; Joseph, J M

1986-01-01

131

Quantification of cyclosporine and tacrolimus in whole blood. Comparison of liquid chromatography–electrospray mass spectrometry with the enzyme multiplied immunoassay technique  

Microsoft Academic Search

ObjectivesThe aim of this work was to compare a validated liquid chromatography–mass spectrometry (LC–MS) method with the commercial enzyme multiplied immunoassay technique (EMIT) for cyclosporine and tacrolimus whole blood quantification.

Nicolas Ansermot; Marc Fathi; Jean-Luc Veuthey; Jules Desmeules; Serge Rudaz; Denis Hochstrasser

2008-01-01

132

Development of Magnetic Particle-based Chemiluminescence Enzyme Immunoassay for the Detection of 17?-estradiol in Environmental Water  

Microsoft Academic Search

In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17?-estradiol (E2) in environmental\\u000a water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix\\u000a and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol–bovine\\u000a serum albumin. The specificity of the anti-E2 antibody

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

2009-01-01

133

An enzyme linked immunoassay for the determination of deoxynivalenol in wheat based on chicken egg yolk antibodies  

Microsoft Academic Search

An indirect competitive enzyme linked immunoassay (ELISA) for the detection of the Fusarium mycotoxin deoxynivalenol (DON) in wheat was developed. Instead of the much more common antibody isolation from mammal serum,\\u000a DON specific antibodies were, for the first time, isolated from the eggs of previously immunized hens. The limit of detection\\u000a was 2 ?g\\/L for standard curves and spiked wheat

L. Schneider; H. Pichler; R. Krska

2000-01-01

134

Development of an Enzyme Immunoassay for the Determination of the Herbicide Metsulfuron-Methyl Based on Chicken Egg Yolk Antibodies  

Microsoft Academic Search

The development of an indirect competitive enzyme immunoassay for the sulfonylurea herbicide metsulfuron-methyl (MSM) is described. In contrast to traditional antibody generation in mammals, this extremely sensitive method is based on chicken egg yolk antibodies (IgY). They were raised in laying hens using an MSM-derivative-BSA hapten as immunogen. With a 1:10000 dilution of the antibody solution and a coating antigen

Elvira Welzig; Harald Pichler; Rudolf Krska; Dietmar Knopp; Reinhard Niessner

2000-01-01

135

Comparison of Four Enzyme Immunoassays for Detection of Human T-Cell Lymphotropic Virus Type 2 Antibodies  

Microsoft Academic Search

Four licensed enzyme immunoassay (EIA) kits for the measurement of antibody to human T-cell lympho- tropic virus (HTLV) type 1, one from Organon Teknika Corp. (OTC), one from Cambridge Biotech Corp. (CBC),andtwofromAbbottLaboratories(the1993modification(Abb93)andthe2.0versionlicensedin1995 (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

DANA GALLO; ELAINE T. YEH; ELLEN S. MOORE; ANDCARL V. HANSON

136

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

Microsoft Academic Search

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to\\u000a control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein\\u000a from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on\\u000a 96- or 384-well microtiter plates in

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

2006-01-01

137

Quantitation of bcl-2 protein in bladder cancer tissue by enzyme immunoassay: comparison with Western blot and immunohistochemistry  

Microsoft Academic Search

Apoptosis (programmed cell death) and the genes regu- lating this process (e.g., bcl-2), have recently become a focus of interest in the study of cancer development and progression. We adapted and evaluated a new enzyme immunoassay method (EIA) for quanitifying bcl-2 in cell lysates. The range of detection of the assay was 5- 400 kilounits\\/L with inter- and intraassay CVs

Sanaa Eissa; Laila S. Seada

1998-01-01

138

Analysis of Mulloidichythys auriflamma for Ciguatera by the Stick Enzyme Immunoassay, Guinea?pig Atrial and Mouse Assays  

Microsoft Academic Search

This study presents chemical, pharmacological and immunological data of marine toxin(s) in the gut and flesh of Mulloidichythys auriflamma (weke). Forty?four per cent of weke tested gave a negative result with the stick enzyme immunoassay (S?EIA), while 56% were in the toxic category. Extracts of gut and flesh of the positive S?EIA samples gave high values of 1193 and 1364

Y. Hokama; A. Y. Asahina; K. Katsura; E. Shang; J. T. Miyahara

1990-01-01

139

European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples?  

PubMed Central

A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

Gray, Jim J.; Kohli, Evelyne; Ruggeri, Franco M.; Vennema, Harry; Sanchez-Fauquier, Alicia; Schreier, Eckart; Gallimore, Chris I.; Iturriza-Gomara, Miren; Giraudon, Helene; Pothier, Pierre; Di Bartolo, Ilaria; Inglese, Nadia; de Bruin, Erwin; van der Veer, Bas; Moreno, Silvia; Montero, Vanessa; de Llano, Mari C.; Hohne, Marina; Diedrich, Sabine M.

2007-01-01

140

Effect of patient characteristics on performance of an enzyme immunoassay for detecting cervical Chlamydia trachomatis infection.  

PubMed Central

We compared the performance of a commercial enzyme immunoassay (EIA) (Chlamydiazyme; Abbott Diagnostics, North Chicago, Ill.) with that of cell culture for the detection of Chlamydia trachomatis cervical infection in 1,417 women attending public health clinics. Confirmatory chlamydial testing by a direct fluorescent-antibody test (MicroTrak; Syva Co., Palo Alto, Calif.) was performed on specimens from women who had positive EIAs. Overall, only 57% of women who had a positive chlamydial test by cell culture were also positive by EIA. We noted a strong association between the number of chlamydial inclusions in cell culture and a positive EIA outcome. The proportion of culture-positive women who also had a positive EIA declined with age and a history of previous sexually transmitted disease and increased among oral contraceptive users. The results of direct fluorescent-antibody confirmatory testing suggested that cell culture was also insensitive for the detection of C. trachomatis infection. Our observations demonstrate that the performance of the chlamydial EIA may vary greatly with individual patient characteristics and that the utility of EIA as a screening test may be limited, especially in older women.

Magder, L S; Klontz, K C; Bush, L H; Barnes, R C

1990-01-01

141

[Enzyme immunoassay of the secondary metabolites of micromycetes as components of lichen substances].  

PubMed

The composition of low-molecular biologically active metabolites typical of microscopic fungi has been studied in blastemas of fruticose lichens of the genera Cladonia, Cetraria, Evernia, Bryoria, and Usnes. The enzyme immunoassay method showed the presence of sterigmatocystin, emodin, mycophenolic acid, citrinin, alternariol, and diacetoxyscirpenol, which occurred regularly and, in most cases, at a frequency of 55 to 100%. The highest levels of accumulation were 0.001-0.003% for emodin, 0.0002% for alternariol and citrinin, 0.0001% for sterigmatocystin and mycophenolic acid, and 0.00005% of the weight of air-dry material for diacetoxyscirpenol. Other metabolites (cyclopiazonic acid, ergot alkaloids, ochratoxin A, PR toxin, deoxynivalenol, zearalenone, and fumonisins) were detected in these lichens less frequently (sometimes only upon the expansion of the territory of sampling), and their content was no more than 0.00005%. The peculiarities of the component composition and the levels of accumulation of fungal metabolites in lichens of different taxonomic affiliation were discussed. PMID:22567889

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

142

Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria  

PubMed Central

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

143

Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay.  

PubMed

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively. PMID:9692222

Yoon, S R; Nah, J J; Kim, S K; Kim, S C; Nam, K Y; Jung, D W; Nah, S Y

1998-07-01

144

Preparation of monoclonal antibody against ginsenoside Rf and its enzyme immunoassay.  

PubMed

A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a K chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids. PMID:10823656

Nah, J J; Song, J Y; Choi, S; Kim, S C; Rhim, H W; Oh, T H; Lee, S M; Nah, S Y

2000-05-01

145

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

NASA Astrophysics Data System (ADS)

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

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

1994-03-01

146

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

PubMed Central

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

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

1997-01-01

147

Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.  

PubMed Central

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.

Kunakorn, M; Markham, R B

1995-01-01

148

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

149

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.

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

2003-01-01

150

Estrogen & progesterone receptors measurement in breast cancer with enzyme-immunoassay & correlation with other prognostic factors.  

PubMed

Estrogen and progesterone receptor (ER and PgR) estimation was carried out by an enzyme-immunoassay by a 'sandwich' technique using two different monoclonal antibodies against each receptor on 508 consecutive breast cancer samples. 43.9 per cent of the tumours were ER+ve and 26.6 per cent were PgR+ve; 23.8 per cent were both ER and PgR+ve, 53.3 per cent were both ER and PgR-ve, 20.0 per cent were ER+ve PgR-ve and 2.8 per cent were ER-ve PgR+ve. Both ER and PgR positivity was associated with increasing age, and this was seen within both pre and post-menopausal subgroups. Grades I and II tumours were more often ER and PgR+ve compared with grade III tumours, indicating that receptor positivity is a marker of a more well differentiated tumour phenotype. Receptor positivity was higher in primary tumours compared to that in metastatic tissues. The proportion of tumours that was ER+ve was found to vary among the four major religious communities, viz., Hindu, Muslim, Christian and Parsi, and this variation was significant in the overall analysis (P less than 0.01). The Christians had the highest rate of ER+ve tumours while the Muslims had the lowest rate. No correlation was observed between ER and PgR status and axillary nodal involvement or tumour size, suggesting that ER and PgR are independent prognostic factors in breast cancer. We found the EIA method to be an easy and rapid technique for ER and PgR analysis and which requires a small amount of tissue and does not involve the use of radioisotopes. PMID:1597324

Redkar, A A; Kabre, S S; Mittra, I

1992-02-01

151

Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A.  

PubMed Central

The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study. Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells. The tubes and plates were examined at 24 and 48 h for cytotoxicity. Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C. difficile were used to evaluate discrepant results. Of 504 samples, 66 were positive and 409 were negative by both tests. Eight samples had indeterminate PTA EIA results and were excluded from this analysis. Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive. Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC. The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA. The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA. We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B.

De Girolami, P C; Hanff, P A; Eichelberger, K; Longhi, L; Teresa, H; Pratt, J; Cheng, A; Letourneau, J M; Thorne, G M

1992-01-01

152

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

PubMed

We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1-30ngmL(-1) with the detection limit of 0.1ngmL(-1). The linear detection range could match the concentration range within the "diagnostic gray zone" of serum f-PSA levels (4-10ngmL(-1)). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4ngmL(-1)). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum. PMID:24139579

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

2013-09-28

153

Evaluation of an Enzyme Immunoassay for Detection of Histoplasma capsulatum Antigen from Urine Specimens.  

PubMed

Detection of Histoplasma capsulatum urinary antigen (UAg) is important for the initial diagnosis of infection and for monitoring of patient responses to antifungal therapy. This study evaluated an analyte-specific reagent (ASR) enzyme immunoassay (EIA) for the detection of H. capsulatum UAg from Immuno Mycologics, Inc. (IMMY) (Norman, OK) in comparison with routine testing with the MiraVista (MVista) H. capsulatum quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Using prospectively collected urine specimens (n = 1,003), we observed an overall percent agreement between the two assays of 97.6% (979/1,003 samples). Compared with the MVista EIA, the sensitivity and specificity of the IMMY ASR EIA were 64.5% (40/62 samples) and 99.8% (939/941 samples), respectively, using a cutoff value of 0.5 ng/ml. Based on available clinical histories for 23/24 discordant samples, 5 IMMY assay-negative/MVista assay-positive samples were considered falsely positive. Furthermore, 10/23 discordant samples were positive by the MVista EIA but were below the limit of quantitation (<0.4 ng/ml). The clinical significance of these low positive results in the MVista EIA is unclear. In addition to the prospective study, we tested 11 urine specimens collected from patients with culture-confirmed Histoplasma infections, and 100% (11/11 samples) were positive by the IMMY ASR EIA. In conclusion, the IMMY ASR EIA may offer an alternative approach for the detection of Histoplasma UAg. Additional prospective studies are needed to better characterize the performance of the IMMY ASR EIA in conjunction with clinical and laboratory findings. PMID:23966508

Theel, Elitza S; Jespersen, Deborah J; Harring, Julie; Mandrekar, Jay; Binnicker, Matthew J

2013-08-21

154

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

2006-02-21

155

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor  

Microsoft Academic Search

A rapid biosensor assay procedure that utilizes biotin–streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-?l sample were approximately

William E Lee; H. Gail Thompson; John G Hall; Douglas E Bader

2000-01-01

156

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

PubMed

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis. PMID:20223258

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

2010-03-16

157

Comparison of Diagnostic Sensitivities of Three Assays (Bartels Enzyme Immunoassay (EIA), Biotest EIA, and Binax NOW Immunochromatographic Test) for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine  

Microsoft Academic Search

The Bartels enzyme immunoassay (EIA), Biotest EIA, and Binax NOW immunochromatographic test (ICT) urinary antigen kits for the detection of Legionella pneumophila serogroup 1 were compared using 178 frozen urine samples. When nonconcentrated urine samples were used, the sensitivity levels of both enzyme EIAs were significantly higher than the sensitivity level of the ICT (Bartels EIA, 71.3%; Biotest EIA, 65.1%;

Carmen Guerrero; Carmen M. Toldos; Genoveva Yague; Cristobal Ramírez; Tomas Rodríguez; Manuel Segovia

2004-01-01

158

Determination of natural olive fruit fly pheromone in insect samples by enzyme linked immunoassays  

Microsoft Academic Search

The olive fruit fly pheromone avidin–biotin ELISA immunoassay, based on the use of polyclonal G antibodies derived from rabbits (reported previously) and a newer assay, based on the use of polyclonal Y antibodies isolated from the eggs of laying hens (reported in this paper), were applied successfully for the analysis of natural pheromone in virgin adult female olive fruit flies.

Afroditi Neokosmidi; Valentine Ragoussis; Christos Zikos; Maria Paravatou-Petsotas; Evangelia Livaniou; Nikitas Ragoussis; Gregory Evangelatos

2008-01-01

159

Enzyme Immunoassay for 2,4,6-Trichloroanisole Based on Monoclonal Antibodies  

Microsoft Academic Search

Monoclonal antibodies (mabs) are of great interest according to their homologous abilities, high specificity, and unlimited possibility of production. Mabs against different substances have been developed. They are used in many fields of application (e.g., medicine and pesticide analysis). Immunoassays performed with mabs have the advantages of easy handling and high sensitivity. The goal of this study was to develop

Ralph Lausterer; Nuria Sanvicens; M. Pilar Marco; Bertold Hock

2003-01-01

160

Analytical performance of a sandwich enzyme immunoassay for prebeta1HDL in stabilized plasma  

Microsoft Academic Search

We have established an immunoassay for pre ? 1- HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre ? 1-HDL is un- stable during storage, fresh plasma must be used for pre ? 1- HDL measurements. In this study, we describe a method of stabilizing pre ? 1-HDL, and evaluate the analytical perfor- mance of

Takashi Miida; Osamu Miyazaki; Yasushi Nakamura; Satoshi Hirayama; Osamu Hanyu; Isamu Fukamachi; Masahiko Okada

2003-01-01

161

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

162

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.

DeLuca, Jane

2007-01-01

163

Subpicogram Determination of Oxytocin by an Enzyme Immunoassay Using Acetylcholinesterase as Label  

Microsoft Academic Search

The pure tetrameric form of Acetylcholinesterase (EC-3.1.1.7) from the electric eel electrophorus electricus has been covalendy coupled to oxytocin. This conjugate has been used as tracer in a heterologous competitive immunoassay. Microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody were used to separate bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was

P. G. Marnet; H. Volland; P. Pradelles; J. Grassi; M. Beaufils

1994-01-01

164

Comparison of nonspecific reactivity in indirect and reverse immunoassays for measles and mumps immunoglobulin M antibodies.  

PubMed Central

Serum specimens collected from patients convalescing from acute measles or mumps infections, other viral infections, or rheumatoid arthritis and from blood donors were tested in indirect and reverse assays for measles and mumps immunoglobulin M (IgM) antibodies. All the samples from patients convalescing from acute mumps and measles infections gave positive IgM results in both tests. However, 6% of sera from patients recovering from other viral infections, 68.4% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive results by the indirect measles IgM enzyme immunoassay (EIA). By the indirect mumps IgM EIA, 9% of sera from other viral infections, 70.1% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive reactions. The reverse test system for measles IgM gave false-positive results in 1.5% of sera from the group with other viral infections, and the reverse mumps EIA gave false-positive results in 0.9% of the patients. Other sera groups did not react in either measles or mumps reverse IgM assays. The results indicated that although nonspecific reactions are frequent in indirect IgM tests for viral antibodies, such reactions are rarely encountered when reverse IgM EIA tests are employed.

Tuokko, H

1984-01-01

165

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

PubMed

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g. PMID:15164837

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

166

Detection of Chlamydia trachomatis in Urine Samples by Nucleic Acid Tests: Comparison with Culture and Enzyme Immunoassay of Genital Swab Specimens  

Microsoft Academic Search

Two commercially available nucleic acid-based tests, ligase chain reaction (LCR; Abbott Laboratories) and PCR (Roche Diagnostics), for the detection of Chlamydia trachomatis in male and female urine samples were compared with culture and enzyme immunoassay (EIA) (Microtrak; Syva) for C. trachomatis detection in genital samples. The samples were collected from 1,005 patients who attended a sexually transmitted disease clinic. In

SONIA SCHEPETIUK; TUCKWENG KOK; LEANNE MARTIN; RUSSELL WADDELL; GEOFFREY HIGGINS

1997-01-01

167

Comparison of a Commercial Reversed Passive Latex Agglutination Assay to an Enzyme Immunoassay for the Detection of Shiga Toxin-Producing Escherichia coli  

Microsoft Academic Search

A multicenter study was performed to compare the performance of a prototypic reversed passive latex agglutination assay (VTEC Screen “Seiken”; Denka-Seiken, Japan) with the Premier EHEC Enzyme Immunoassay (Meridian Diagnostics, USA) for the detection of Shiga toxin in 554 diarrheal stool samples. Standard culture on sorbitol MacConkey agar and the use of latex agglutination reagents were included to identify the

K. C. Carroll; K. Adamson; K. Korgenski; A. Croft; R. Hankemeier; J. Daly; C. H. Park

2003-01-01

168

Comparison of DNA Enzyme Immunoassay and Line Probe Assays (Inno-LiPA HCV I and II) for Hepatitis C Virus Genotyping  

Microsoft Academic Search

Two methods for genotyping hepatitis C virus (DNA enzyme immunoassay (DEIA) and line probe assay (Inno-LiPA HCV I and II)) were compared on 120 samples and of these 87% were assigned to the same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the

S. LE POGAM; F. DUBOIS; R. CHRISTEN; C. RABY; A. CAVICCHINI; A. GOUDEAU

1998-01-01

169

Quantification of polymerase chain reaction products: enzyme immunoassay based systems for digoxigenin- and biotin-labelled products that quantify either total or specific amplicons  

Microsoft Academic Search

Enzyme immunoassays were developed for quantification of polymerase chain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to

J. Stevens; F. S. Yu; P. M. Hassoun; J. J. Lanzillo

1996-01-01

170

Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women  

Microsoft Academic Search

OBJECTIVE: To evaluate the commercial microparticle enzyme immunoassay (MEIA), Abbott IMx Select Chlamydia, for the detection of Chlamydia trachomatis in women and to compare its performance with endocervical cell culture. Also, to determine whether sampling the urethral site is an important part of chlamydial diagnosis in women. SETTING: The Auckland, Manukau, and Waitakere Sexual Health Clinics, Auckland, New Zealand and

M K Brokenshire; P J Say; A H van Vonno; C Wong

1997-01-01

171

Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella sp. and Yersinia enterocolitica O:9 in cattle and pigs  

Microsoft Academic Search

A rapid, inexpensive and rugged serological test that distinguishes cattle and swine infected with Brucella sp. or Yersinia enterocolitica O:9 is described. The test protocol, which is an indirect enzyme immunoassay uses a high concentration of divalent cation chelating agents to minimize binding of Y. enterocolitica O:9 antibody to rough lipopolysaccharide antigen derived from B. abortus RB51. No false positive

K. Nielsen; P. Smith; W. Yu; P. Nicoletti; G. Jungersen; J. Stack; J. Godfroid

2006-01-01

172

Comparison of DNA probe, monoclonal antibody enzyme immunoassay, and cell culture for the detection of Chlamydia trachomatis.  

PubMed Central

A total of 201 endocervical specimens were obtained from patients with a clinical or epidemiological history suggestive of chlamydial infection. These specimens were tested by DNA probe (Gen-Probe, San Diego, Calif.) and the IDEIA III (Boots-Celltech, Berkshire, United Kingdom) monoclonal antibody enzyme immunoassay and compared with cell culture for detection of Chlamydia trachomatis. Discrepancies between cell culture and antigen detection methods were resolved by direct fluorescent-antibody testing. In a population with a 17.4% prevalence, the sensitivities and specificities of these assays were 82.8 and 99.4%, respectively, for the DNA probe assay and 97.1 and 98.1%, respectively, for the IDEIA III.

LeBar, W; Herschman, B; Jemal, C; Pierzchala, J

1989-01-01

173

Reverse transcription-PCR detection of LaCrosse virus in mosquitoes and comparison with enzyme immunoassay and virus isolation.  

PubMed

A reverse transcription-PCR (RT-PCR) assay was developed and compared with enzyme immunoassay (EIA) and virus isolation for detecting LaCrosse virus (LAC) in mosquito pools. All three techniques were able to detect a single LAC-infected mosquito in a pool of 99 negative mosquitoes. Virus isolation was the most sensitive of the three techniques; it was possible to isolate virus immediately following intrathoracic inoculation of mosquitoes. RT-PCR was second in sensitivity; LAC RNA was detected 1 day postinfection. EIA detected LAC antigen 2 days postinfection. Additionally, RT-PCR and EIA were able to detect LAC RNA and protein, respectively, from mosquito samples which were subjected to seven freeze-thaw cycles, and RT-PCR was able to detect LAC RNA from mosquito samples which remained at room temperature for up to 7 days. PMID:7814527

Wasieloski, L P; Rayms-Keller, A; Curtis, L A; Blair, C D; Beaty, B J

1994-09-01

174

A novel protease activity assay method based on an engineered autoinhibited protein using an enzyme-linked immunoassay.  

PubMed

Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification. PMID:24106734

Yoon, Hyun Kyung; Yoo, Tae Hyeon

2013-10-08

175

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

176

Quantification of domoic acid in shellfish samples by capillary electrophoresis-based enzyme immunoassay with electrochemical detection.  

PubMed

A new method was developed to quantify domoic acid by capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection. The method was based on noncompetitive immunoreaction between free domoic acid antigen (Ag) and excessive amount of horseradish peroxidase (HRP)-labeled antidomoic acid antibody tracer (Ab*) in liquid phase. Then the bound enzyme-labeled complex (Ab*-Ag) and unbound Ab* were separated by CE, and the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted for EC detection. Using CE-EIA with EC detection, equilibrium was reached in 30 min, and the analytical results were obtained within a further 5 min. The linear range and the detection limit were 0.1-50 ng/mL and 0.02 ng/mL, respectively. Analytes recoveries were 89.6-105.8%. The sensitivity of the method was 16 times greater than that of enzyme-linked immunosorbent assay (ELISA). The developed method was applied to quantitatively analysis of domoic acid in contaminated shellfish samples with rapid and simple pretreatment, and the results were consistent with the same samples analyzed through ELISA. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for determination of domoic acid in shellfish samples. PMID:22406515

Zhang, Xiao-Wei; Zhang, Zhao-Xiang

2012-03-01

177

IgM, IgG and IgA class enterobacterial antibodies in serum and synovial fluid in patients with ankylosing spondylitis and rheumatoid arthritis  

Microsoft Academic Search

SUMMARY IgM, IgG and IgA class antibodies against three Klebsiella pneumoniae capsular types, Escherichia coli and Proteus mirabilis, as well as total immunoglobulin concentrations, were measured by enzyme immunoassay and radial immunodiÄusion tech- nique, respectively, in paired serum and synovial fluid samples from eight patients with ankylosing spondylitis and 10 with rheumatoid arthritis. No clear evidence for intra-articular antibody production

O. MAKI-IKOLA; M. PENTTINEN; R. VON ESSEN; C. GRIPENBERG-LERCHE; H. ISOMAKI; K. GRANFORS

1997-01-01

178

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

179

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.

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

2011-01-01

180

Cross-reactivity of non-Aspergillus fungal species in the Aspergillus galactomannan enzyme immunoassay.  

PubMed

The Aspergillus galactomannan enzyme-linked immunosorbant assay (EIA) has been demonstrated to facilitate rapid and sensitive detection of invasive aspergillosis. However, test specificity has not been fully evaluated in non-Aspergillus fungal species. Of 53 fungal isolates, cross-reactivity was observed with 5 non-Aspergillus spp.: Blastomyces dermatitidis, Nigrospora oryzae, Paecilomyces lilacinus, Penicillium chrysogenum, and Trichothecium roseum. PMID:17662550

Cummings, Jessica R; Jamison, Ginger R; Boudreaux, Jan W; Howles, Merry J; Walsh, Thomas J; Hayden, Randall T

2007-07-26

181

Detection of Serum Transforming Growth Factor-? in Patients of Primary Epithelial Ovarian Cancers by Enzyme Immunoassay  

Microsoft Academic Search

Transforming growth factor-? (TGF-?) is a potent mitogenic polypeptide. It is secreted by a variety of transformed cells and tumors, modifying tumor growth through autocrine or paracrine mechanism. In the present study, serum levels of TGF-? were determined by enzyme-linked immunosorbent assay (ELISA) in 27 normal females, 116 patients with benign ovarian tumors, and 42 patients with epithelial ovarian cancers

Chin-Hsiang Chien; Chien-Chu Huang; Yu-Hung Lin; Jenta Shen; Song-Nan Chow

1997-01-01

182

Performance of the TechLab C. DIFF CHEK-60 Enzyme Immunoassay (EIA) in Combination with the C. difficile Tox A\\/B II EIA Kit, the Triage C. difficile Panel Immunoassay, and a Cytotoxin Assay for Diagnosis of Clostridium difficile-Associated Diarrhea  

Microsoft Academic Search

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A\\/B II enzyme immunoassay (Tox-A\\/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile

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

2004-01-01

183

Rapid confirmation of polymyxin-cloth enzyme immunoassay for group D salmonellae including Salmonella enteritidis in eggs by polymerase chain reaction  

Microsoft Academic Search

A rapid, simple and inexpensive combined screening test and confirmation procedure was developed for group D Salmonellae (e.g. Salmonella enteritidis) in eggs. S. enteritidis inoculated into egg samples was pre-enriched in a minimal volume of enrichment broth, which was then assayed by the polymyxin-cloth enzyme immunoassay (polymyxin-CEIA): cholate-extracted lipopolysaccharide antigens were captured on polymyxin-coated polyester cloth and detected by sequential

Haiyan Wang; Burton W. Blais; Hiroshi Yamazaki

1995-01-01

184

A three-parameter langmuir-type model for fitting standard curves of sandwich enzyme immunoassays with special attention to the ?-fetoprotein assay  

Microsoft Academic Search

In a simplified approach to the reaction kinetics of enzyme-linked immunoassays, a Langmuir-type equation y = [ax\\/(b + x)] + c was derived. This model proved to be superior to logit-log and semilog models in the curve-fitting of standard curves. An assay for ?-fetoprotein developed in our laboratory with a sensitivity of 2 g\\/liter and a between-day coefficient of variation

W. Kortlandt; H. J. Endeman; J. O. O. Hoeke

1987-01-01

185

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

Microsoft Academic Search

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in

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

2008-01-01

186

Comparison of conventional autoradiography with a new DNA enzyme immunoassay for the detection of hepatitis C virus-polymerase chain reaction amplification products  

Microsoft Academic Search

The detection of HCV-PCR amplification products by DNA enzyme immunoassay (DEIA) was compared with conventional hybridization carried out with a 32P-labelled oligonucleotide probe. The detection limit of both methods was shown to be between 100 pg and 1 ng of amplicon. All serum samples of 40 HCV-seropositive patients were positive after PCR in autoradiography, but only 38 with the DELA

H.-H. Feucht; B. Zöllner; R. Laufs

1995-01-01

187

PCR-enzyme immunoassay of rDNA in the diagnosis of candidemia and comparison with amplicon detection by agarose gel electrophoresis  

Microsoft Academic Search

We have developed a semi-nested PCR-enzyme immunoassay (snPCR-EIA) for the detection of Candida species in serum specimens, and the sensitivity of amplicon detection was compared with the detection of amplified product by agarose gel electrophoresis (AGE). The universal outer primers amplified the 3? end of 5.8S and the 5? end of 28S rDNA including the internally transcribed spacer 2 (ITS2)

Suhail Ahmad; Abu S Mustafa; Zaiba Khan; Areej I Al-Rifaiy; Zia U Khan

2004-01-01

188

New Enzyme Immunoassays for Sensitive Detection of Circulating Candida albicans Mannan and Antimannan Antibodies: Useful Combined Test for Diagnosis of Systemic Candidiasis  

Microsoft Academic Search

Two standardized enzyme immunoassays for the serological diagnosis of candidiasis were developed. The first one detects antimannan antibodies, while the second one detects mannan with a sensitivity of 0.1 ng\\/ml. These tests were applied to 162 serum samples retrospectively selected from 43 patients with mycologically and clinically proven candidiasis caused by Candida albicans. Forty-three serum samples were positive for mannan,

BOUALEM SENDID; MARC TABOURET; JEAN LOUIS POIROT; DANIEL MATHIEU; JEANINE FRUIT; DANIEL POULAIN

1999-01-01

189

Relationship of Serum Estradiol and Progesterone Concentrations to the Excretion Profiles of Their Major Urinary Metabolites as Measured by Enzyme Immunoassay and Radioimmunoassay  

Microsoft Academic Search

Paired daily blood and urine samples were collected from 10 apparently healthy premenopausal women to compare the hormone profiles of estradiol (E2) and progesterone in serum with those of estrone conjugates (E,Conj) and pregnanediol-3-glucuronide (PdG) in urine. Serum hor- mones were measured by radioimmunoassay (AlA) kits, whereas the urinary steroid metabolites were assessed by both AlA and enzyme immunoassay (EIA).

C. J. Munro; G. H. Stabenfeldt; J. R. Cragun; L. A. Addiego; J. W. Overstreet; B. L. Lasley

1991-01-01

190

3,3?,5,5?-Tetramethylbenzidine as an Ames Test Negative Chromogen for Horse-Radish Peroxidase in Enzyme-Immunoassay  

Microsoft Academic Search

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

E. S. Bos; A. A. van der Doelen; N. van Rooy; A. H. W. M. Schuurs

1981-01-01

191

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay  

Microsoft Academic Search

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a

E. W. Weiler; P. S. Jourdan; W. Conrad

1981-01-01

192

Prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae Immunoglobulin G and A Antibodies in a Healthy Finnish Population as Analyzed by Quantitative Enzyme Immunoassays  

Microsoft Academic Search

Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G (IgG) and IgA antibody seropreva- lence rates and antibody levels related to age and gender were studied. The samples (n 5 742) were collected during a nonepidemic period and analyzed by quantitative enzyme immunoassays (EIAs). Seroprevalence to C. pneumoniae was found to increase sharply in young children, and in the 15- to 19-year-old

TAMARA TUUMINEN; SIRPA VARJO; HEIDI INGMAN; THEODOR WEBER; JARMO OKSI; MATTI VILJANEN

2000-01-01

193

Determination of CD59 protein in normal human serum by enzyme immunoassay, using octyl-glucoside detergent to release glycosyl-phosphatidylinositol-CD59 from lipid complex  

Microsoft Academic Search

In this study we have optimised the enzyme immunoassay (ELISA) to quantify CD59 antigen in human serum or plasma. The glycosyl-phosphatidylinositol (GPI)-linked form of CD59 is known to complex with serum high-density lipoprotein. For ELISA optimisation, therefore, we investigated the effect of detergents, added to the sample diluent, on the determined values of CD59. Values obtained in the presence of

A. P. G Landi; A. B Wilson; A Davies; P. J Lachmann; V. P. L Ferriani; D. J Seilly; A. I Assis-Pandochi

2003-01-01

194

Oestrogen and progesterone receptor estimation by enzyme-immunoassay on tissues removed before and after a modified radical mastectomy.  

PubMed

To investigate the possibility of receptor degradation due to devascularization of the tumour during mastectomy, oestrogen and progesterone receptors (ER and PgR) were measured by an enzyme-immunoassay (EIA) in 59 cases of primary breast cancer on samples taken before and after performing a modified radical mastectomy. Pre- and post-mastectomy samples from the same patient were analysed simultaneously in the same assay run. There was 86.4% and 93.2% agreement respectively in ER and PgR status between samples removed before and after surgery. When actual values were analysed, the post-mastectomy values were higher or lower than pre-mastectomy values with similar frequency. These random variations could be attributed to heterogeneous distribution of receptors within a tumour. The overall correlation between pre- and post-mastectomy values was excellent (ER: r = 0.810, P < 0.001; PgR: r = 0.706, P < 0.001). Devascularization of the tumour during surgery does not seem to affect the integrity of the epitopes recognized by monoclonal antibodies against ER and PgR to any significant extent. PMID:8359284

Vyas, J J; Redkar, A A; Kabre, S S; Mittra, I

1993-08-01

195

Comparison of the PREMIER cryptococcal antigen enzyme immunoassay and the latex agglutination assay for detection of cryptococcal antigens.  

PubMed Central

A new enzyme immunoassay (EIA), PREMIER Cryptococcal Antigen, was compared with latex agglutination (LA) for the detection and quantitation of circulating capsular polysaccharide antigen from Cryptococcus neoformans. The clinical evaluation of PREMIER EIA as a screening assay, including 475 specimens with 120 LA and EIA positives, resulted in 99% sensitivity and 97% specificity. The clinical specimens included sera and cerebrospinal fluids as well as 10 rheumatoid factor-positive and 20 anti-nuclear antibody-positive serum samples. This monoclonal antibody-based assay detects serotypes A to D at 0.63, 0.63, 7.8, and 62 ng/ml, respectively. With three different known positive specimens, the assay was found to yield coefficients of variation of 2 to 12% for intra- and interassay comparisons of precision and reproducibility. The primary use for semiquantitative values derived with the LA or EIA is to follow the course of disease and monitor drug therapies. The present data suggest that the PREMIER EIA will be a valuable method for this purpose. We conclude that the PREMIER Cryptococcal Antigen EIA provides a rapid, convenient, and reliable antigen detection method for screening and semiquantitative determination of antigen levels.

Gade, W; Hinnefeld, S W; Babcock, L S; Gilligan, P; Kelly, W; Wait, K; Greer, D; Pinilla, M; Kaplan, R L

1991-01-01

196

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

PubMed

Measurement of free plasma metanephrines (metanephrine and normetanephrine), usually performed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), has been recommended as the single biochemical test of choice for the diagnosis of pheochromocytoma. Alternatively, a widely available, simple means to measure these biomarkers with enzyme immunoassay (EIA) needs to be studied. The aim of this study was to investigate the diagnostic efficacy of such a method in comparison with (131)I-metaiodobenzylguanidine (MIBG) whole body scan (WBS) in patients with pheochromocytoma. We enrolled patients undergoing (131)I-MIBG WBS due to clinical findings suggestive of pheochromocytoma (n = 45), and patients with primary hypertension (n = 36). All subjects had blood tests for free plasma metanephrine (MN) and normetanephrine (NM) with a commercially available EIA kit. WBS was positive in 30 pheochromocytoma patients and negative in 15 refuted ones, with 100% accuracy. The sensitivity, specificity and accuracy of MN and NM in combination (either or both positive) were 96.7%, 86.3% and 90.1%, showing comparable diagnostic performance both to (131)I-MIBG WBS (all p > 0.1), and also to the same markers measured with HPLC-ECD reported in the literature. These results showed that the EIA method may be eligible as an alternative to HPLC-ECD for plasma metanephrine determination in the identification of pheochromocytoma. PMID:18618218

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

2008-07-11

197

Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay.  

PubMed Central

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.

Dennehy, P H; Gauntlett, D R; Spangenberger, S E

1990-01-01

198

Assessment of Aspergillus fumigatus Burden in Pulmonary Tissue of Guinea Pigs by Quantitative PCR, Galactomannan Enzyme Immunoassay, and Quantitative Culture?  

PubMed Central

Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log10 higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.

Vallor, Ana C.; Kirkpatrick, William R.; Najvar, Laura K.; Bocanegra, Rosie; Kinney, Marsha C.; Fothergill, Annette W.; Herrera, Monica L.; Wickes, Brian L.; Graybill, John R.; Patterson, Thomas F.

2008-01-01

199

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

PubMed Central

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

Uslan, Daniel Z.; Rubin, Zachary

2013-01-01

200

Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes).  

PubMed

Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three-fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies. PMID:21538448

Heintz, Matthew R; Santymire, Rachel M; Parr, Lisa A; Lonsdorf, Elizabeth V

2011-05-02

201

Comparison of Antinuclear Antibody Testing Methods: Immunofluorescence Assay versus Enzyme Immunoassay  

Microsoft Academic Search

Performances of anti-nuclear antibody testing by immunofluorescence assay (ANA-IFA) and enzyme immu- noassay (ANA-EIA) were compared in relation to patient diagnosis. A total of 467 patient serum samples were testedbyANA-IFA(Kallestad;Sanofi)andANA-EIA(RADIAS;Bio-Rad),andtheirage,sex,diagnosis,disease status, and medications were obtained through chart review. Reference ranges were established by testing 98 healthy blood donor samples. Eighty-six samples came from patients with diffuse connective tissue diseases, including

RICHARD A. GNIEWEK; DANIEL P. STITES; THOMAS M. MCHUGH; JOAN F. HILTON; ANDMAYUMI NAKAGAWA

1997-01-01

202

Preparation of antibodies and development of an enzyme immunoassay for determination of atrazine in environmental samples  

Microsoft Academic Search

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed and optimized for atrazine determination in soil at different depths (0–10, 10–20, and 20–30 cm) before and after 48 h of application, corn shoot and cow milk samples collected from Dina farm, Egypt. This assay was based on a specific polyclonal antibodies (PAb) raised by immunizing New Zealand rabbits with

Kawther S. El-Gendy; Nagat M. Aly; Eman M. Mosallam; Ahmed K. Salama

2011-01-01

203

Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis  

Microsoft Academic Search

An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with

Ana M. Vigliocco; Patricia S. Silva Paulo; Joaquín Mestre; Gabriel C. Briones; Graciela Draghi; Mirta Tossi; Klaus Nielsen

1997-01-01

204

Prognostic Value of Estrogen and Progesterone Receptors Measured by Enzyme Immunoassays in Human Breast Tumor Cytosols  

Microsoft Academic Search

Clinically significant cut-off values to discriminate between receptor- positive and -negative, and the prognostic value of estrogen receptors (ER) and progesterone receptors (PgR) measured by enzyme immunoas- say (EIA) have not yet been established. We have therefore measured ER and PgR by EIA in cytosols from 205 primary breast cancer biopsies. Clinically significant cut-off values (30 fmol\\/mg protein for ER;

John A. Foekens; Henk Portengen; Wim L. J. van Putten; Harry A. Peters; Hendrik L. J. M. Krijnen; Jana Alexieva-Figusch; Jan G. M. Klijn

205

Enzyme immunoassay for macrolide antibiotics: characterization of an antibody to 23-amino-O-mycaminosyltylonolide.  

PubMed Central

An enzyme-linked immunosorbent assay was developed for the detection of macrolide antibiotics by using a polyclonal antibody generated in rabbits immunized with 23-amino-O-mycaminosyltylonolide (23-amino-OMT) covalently linked to keyhole limpet hemocyanin. The specificity and sensitivity of this antibody were characterized by using 23-amino-OMT coupled to alkaline phosphatase as an enzyme-linked label in a direct competitive enzyme-linked immunosorbent assay. The assay sensitivity was as low as 0.3 ng/ml for 23-amino-OMT, with a 50% inhibitory concentration of 8 ng/ml. This antibody exhibited good reactivity with 12-, 14- or 16-membered macrolides possessing amino-substituted sugar moieties, regardless of the presence of neutral sugar residues. Little or no cross-reactivity was observed with the macrocyclic lactone ring structure (tylactone) or macrolides containing only neutral sugars. No cross-reaction was observed with polyenes or nonmacrolide antibiotics. Known macrolide-producing cultures grown in fermentation broth also showed good reactivity, indicating that this assay is useful in detecting this class of metabolites in fermentation.

Yao, R C; Mahoney, D F

1989-01-01

206

Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.  

PubMed Central

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories. Images

Clem, T R; Yolken, R H

1978-01-01

207

Competitive enzyme immunoassays for the rapid detection of antibodies to feline infectious peritonitis virus polypeptides.  

PubMed Central

Monoclonal antibodies specific for the envelope (E1), peplomer (E2), and nucleocapsid (N) polypeptides of feline infectious peritonitis virus (FIPV) were used in rapid, competitive enzyme-linked immunosorbent assays (ELISA) to study the humoral immune response of cats to FIPV infection. Results from the competitive ELISAs were correlated with those from immunofluorescent antibody assays (IFAs) on 203 samples obtained from 64 individual cats. The IFA results correlated best with those obtained with the anti-E1 specific competitive ELISA (85.7%). In contrast, anti-N and anti-E2 competitive ELISA results correlated with IFA results only 65.5 and 2.4% of the time, respectively. The results of the anti-E1 specific competitive ELISA were not influenced by the total immunoglobulin concentration or the possible presence of free viral antigens in the serum. These results suggest that a competitive ELISA involving the use of enzyme-conjugated monoclonal antibody to the E1 glycoprotein of FIPV is a simple and rapid replacement for the more cumbersome IFA. Images

Fiscus, S A; Teramoto, Y A; Mildbrand, M M; Knisley, C V; Winston, S E; Pedersen, N C

1985-01-01

208

Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay.  

PubMed Central

The technical complexity of determining the serovar of Chlamydia trachomatis strains has limited the use of serotyping in clinical and epidemiologic studies. We developed a simple method for rapidly serotyping isolates of C. trachomatis by using monoclonal antibodies in a dot-enzyme-linked immunosorbent assay (ELISA) system. Isolates were passaged three to six times in shell vial cultures to greater than 50% monolayer infection, and chlamydial elementary bodies were isolated by sonication and microcentrifugation. Chlamydial antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with C. trachomatis-specific monoclonal antibodies. Bound antibody was detected visually by a color reaction by using peroxidase-conjugated anti-mouse immunoglobulins. This method can be routinely applied to 60 or more specimens concurrently. We compared dot-ELISA serotyping with monoclonal antibody microimmunofluorescence serotyping of 124 clinical C. trachomatis isolates and found that dot-ELISA has sensitivity and serotyping accuracy comparable to that of monoclonal antibody microimmunofluorescence. Images

Barnes, R C; Wang, S P; Kuo, C C; Stamm, W E

1985-01-01

209

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

SciTech Connect

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

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

1987-09-01

210

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance.  

PubMed

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA-BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL(-1), and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74-110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL(-1) for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R2 = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples. PMID:17668189

Wang, Xiang-Hong; Liu, Tao; Xu, Na; Zhang, Yan; Wang, Shuo

2007-08-01

211

Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin B1 in food products.  

PubMed

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for determination of aflatoxin B1 (AFB1) was developed. To improve the assay sensitivity, a mixture of 3-(10'-phenothiazinyl)-propane-1-sulfonate and 4-morpholinopyridine previously optimized by a factorial design was used as enhancer of horseradish peroxidase-induced chemiluminescence. Varying the concentrations of the coating anti-AFB1 antibody and conjugate of AFB1 and horseradish peroxidase the conditions of the chemiluminescent assay were optimized. The values of the detection limit value and dynamic working range of CL-ELISA of AFB1 were 0.0015 ng mL(-1) and 0.003-0.03 ng mL(-1), respectively. It was shown that a dilution of rice and mung beans extracts in 5 and 10 times, respectively, prevented a matrix effect of the food products in CL-ELISA. The recovery values from the spiked samples of rice and mung beans were in the range of 90-104% and 102-117%, respectively. Studying 8 rice and 8 mung beans samples purchased in commercial stores the developed CL-ELISA allowed to find 3 samples (1 rice and 2 mung beans) containing AFB1, the content of AFB1 in one sample being higher than the maximum acceptable level established in the European Community. PMID:23598187

Yu, Feng-Yih; Gribas, Anastasia V; Vdovenko, Marina M; Sakharov, Ivan Yu

2013-01-03

212

[Differentiation of matrix proteins of influenza A viruses using enzyme immunoassay].  

PubMed

Matrix protein is known as a type-specific structural protein of influenza viruses. An attempt has been made to find out whether or not strain-specific components could be detected from matrix protein, in addition to its type-specific antigen determinants. The technique of enzyme immune assay was chosen as the optional method to differentiate between matrix proteins of various influenza-A viruses. Antigen titration was undertaken of several matrix proteins, using two specific anti-matrix-protein sera in each case. Information regarding serological relationships between the tested matrix proteins of various influenza-A viruses was obtained from a quotient between the titres of one antigen, on the one hand, and the two anti-matrix-protein sera used in titration, on the other. Two matrix protein sub-types were established in the context of the influenza-A viruses tested. Sub-type M1 was attributable to older strains (A/PR/8 and A/FM/1), whereas the matrix protein of sub-type M2 was found to be present in more recent strains (A/Hongkong and A/Port Chalmers). PMID:83125

Mohr, C; Döhner, L; Herrmann, B; Herrmann, H

1978-01-01

213

Method of analysis of non-competitive enzyme immunoassays for antibody quantification.  

PubMed

A computerized analysis of a quantitative enzyme-linked immunoadsorbent assay (EIA) using a non-specific immunoglobulin (IgG) of known concentration as the standard has been developed for measuring specific antibody levels in serum without the need for affinity purification of the positive control antibody. The computer program utilized logit-log linear regression analysis of sigmoid serial dilution curves plus a weighted least-squares best curve fit analysis and an iterative manipulation to eliminate errant data points. The EIA was performed using serial dilutions of standard and unknown antibodies, and a double sandwich technique. A comparison of antibody levels determined by EIA using non-specific IgG as a standard relative to antibody levels determined using affinity-purified specific antibody as a standard were 1.04, 0.53, 0.48, and 0.97 for four different polyclonal antibody systems. Five monoclonal antibodies to carcinoembryonic antigen gave ratios as described above of 1.07, 1.59, 1.73, 2.32, and 2.42. The corresponding antibody affinity constants (1/mol) were 1.0 X 10(8), 3.8 X 10(8), 5.5 X 10(9), 1.8 X 10(10), and 2.6 X 10(10) respectively. This method permits accurate quantification of serum antibody levels when affinity-purified antibodies are not readily available and avoids errors due to loss of antibody activity during affinity purification. PMID:3298436

Beatty, J D; Beatty, B G; Vlahos, W G; Hill, L R

1987-06-26

214

Serologic detection of experimental Salmonella enteritidis infections in laying hens by fluorescence polarization and enzyme immunoassay.  

PubMed

Detection of infected poultry flocks is essential for controlling eggborne transmission of Salmonella enteritidis to humans. The present study evaluated the detection of antibodies in the sera of experimentally infected chickens by a fluorescence polarization assay with a tracer prepared from the O-polysaccharide of S. enteritidis and an enzyme-linked immunosorbent assay (ELISA) with an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with either 10(6) or 10(8) colony-forming units (CFU) of S. enteritidis (phage type 13a) or with 10(8) CFU of Salmonella typhimurium. Serum samples were collected before inoculation and at five subsequent weekly intervals. Both assays successfully detected the majority of hens infected with S. enteritidis at either dose level, but they also identified a substantial number of hens infected with S. typhimurium as seropositive. The fluorescence polarization test detected S. enteritidis infection significantly more often and cross-reacted with sera from hens infected with S. typhimurium significantly less often than the ELISA. The fluorescence polarization assay also offered advantages in terms of speed and methodologic simplicity. PMID:11922325

Gast, Richard K; Nasir, Mohammad S; Jolley, Michael E; Holt, Peter S; Stone, Henry D

215

Development of an Enzyme Immunoassay Using a Monoclonal Antibody against the Psychoactive Diterpenoid Salvinorin A.  

PubMed

Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 ?g/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum. PMID:23987562

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

2013-08-29

216

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

PubMed

The geographic distribution and areas of high sylvatic plague activity in California were verified by using coyotes (Canis latrans) as sentinel animals. Antibody levels against Yersinia pestis were tested using the enzyme-labelled antibody (ELA) test and the microtiter passive hemagglutination and hemagglutination inhibition. A survey using the ELA test indicated that the overall antibody prevalence among 143 coyotes was 21%. By geographic regions, the highest antibody prevalence was 27% among coyotes from mountain areas on the northern and eastern borders of the state. This was followed by 19% in the central coastal area and 12% in the central valley. Areas with a high prevalence of seropositive coyotes or high antibody levels in individual coyotes matched the four areas of human plague exposures reported in 1977 and 1978. These areas included the central Sierra mountains adjacent to Lake Tahoe, southeastern Kern County, the central coastal area and Scott Valley near the Oregon border. The ELA test appears to be a promising tool for future epidemiologic studies of plague. PMID:382839

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

1979-09-01

217

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

PubMed

In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda. PMID:19652473

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

2009-07-01

218

Estrogen and progesterone receptors in breast cancer: comparison between enzyme immunoassay and computer-assisted image analysis of immunocytochemical assay.  

PubMed

Evaluation of estrogen (ER) and progesterone (PR) receptor content is now an important procedure in the management of breast cancer patients. Production of monoclonal antibodies to ER and PR has permitted development of an enzyme immunoassay (EIA) and immunocytochemical assay (ICA). This study compared the results of ICA and EIA to evaluate ER and PR in 197 breast cancers using the same monoclonal antibodies. The ICA results were obtained by automated computer-assisted image analysis using CAS 200. The cut-off values adopted were 15 fmol/mg protein for EIA and 10% of the positive neoplastic area of the nuclei for ICA. For statistical analysis, Spearman's correlation coefficient and chi 2 were used. There was good correlation between ICA and EIA for both ER (r = 0.714; p < 0.0001) and PR (r = 0.815; P < 0.0001). Of 197 tumors, 136 (69.04%) were ER-ICA+, and 138 (70.05%) were ER-EIA+; 111 (56.35%) were PR-ICA+, and 115 (58.38%) were PR-EIA+. Results were concordant, positive or negative with both methods, in 175 cases for ER and in 173 cases for PR. ER and PR results were only discordant in 22 and 24 cases, respectively. Concordance of results obtained by the two methods was 88.83% (P < 0.0001) for ER and 87.81% (P < 0.0001) for PR. Correlation of results obtained by EIA and ICA to determine ER and PR was good. The data obtained suggest that ICA with automated image analysis is an effective means for evaluating ER and PR content in human breast cancer, especially when, as happens ever more frequently nowadays, the tumor is too small to perform EIA or when retrospective studies are performed. PMID:8889392

Cavaliere, A; Bucciarelli, E; Sidoni, A; Bianchi, G; Pietropaoli, N; Ludovini, V; Vitali, R

1996-09-15

219

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

PubMed Central

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

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

2005-01-01

220

Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay.  

PubMed Central

We evaluated prospectively the detection of respiratory syncytial virus (RSV) by culture and by direct antigen detection using an indirect immunofluorescence assay (IFA), a direct monoclonal immunofluorescence assay (DFA), and a monoclonal enzyme immunoassay (EIA). Of 221 specimens, 95 (43%) were culture positive for RSV, 4 (1.8%) contained more than one virus, and 17 (7.6%) contained a virus other than RSV. Overall, HEp-2 and Flow 6000 cells grew significantly more RSV isolates (82 and 72%, respectively) than A549 cells, which grew only 29% of the isolates. The mean time for RSV detection with HEp-2 cells was 2.9 days. This was significantly less than the mean time for RSV detection with either Flow 6000 cells (6.1 days) or A549 cells (6.4 days). Of 221 specimens, 129 were tested simultaneously by culture, IFA, and DFA. Of these 129 specimens, 62 (48%) were positive by culture, 69 (53%) were positive by IFA, and 70 (54%) were positive by DFA. For 92 specimens screened simultaneously by culture, IFA, and EIA, positive results were obtained for 33 (36%) of the specimens by both culture and IFA and for 29 (32%) of the specimens by EIA. Of 126 culture-negative specimens, 21 (17%) were positive for RSV when determined by IFA. Conversely, 14 (15%) of 95 RSV culture-positive specimens were negative by IFA, whereas DFA missed 19% of the culture-positive specimens. Compared with culture, the Kallestad EIA kit had a sensitivity and specificity of 73 and 92% respectively, but missed 9 (27%) of 33 culture-positive specimens. These data demonstrate that isolation by culture continues to be important for viral diagnosis of REV infections and that for valid comparative studies between viral isolations and rapid detection methods, both sensitive host cells and appropriate test conditions are required.

Hughes, J H; Mann, D R; Hamparian, V V

1988-01-01

221

A highly sensitive sandwich enzyme immunoassay for human liver-specific antigen (LSA) and its forensic application.  

PubMed

A highly sensitive and specific sandwich enzyme immunoassay for human liver-specific antigen (LSA) was developed and its forensic application using LSA as a marker for the determination of liver injury were examined. The LSA was purified from human liver by immunoaffinity chromatography. Polystyrene ball coated with affinity-purified rabbit anti-human LSA IgG was incubated with the human LSA and then with affinity-purified anti-human LSA Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl) propionic acid as a hydrogen donor. The detection limit for human LSA was 0.52 pg per assay. The serum LSA levels determined by this assay in healthy male and female adults were 1.5-1.6 ng/ml and 0.7-1.0 ng/ml, respectively. The recovery of LSA added to 5 microliters and 10 microliters serum samples was estimated to be 53.1-55.5% and 70.6-74.8%, respectively, and no difference in recovery between serum from males and females was observed. LSA antigenic activity in bloodstains containing LSA was detectable after storage for 30 days at room temperature. High levels of LSA were proved to exist in forensic samples taken from stabbed livers, and it was clearly possible to differentiate between samples from stabbed livers and those originating from other stabbed organs. These findings demonstrate that determination of LSA from forensic samples is useful for detecting liver injuries. PMID:8065064

Seo, Y; Takahama, K

1994-06-01

222

Determination of aflatoxin B1-DNA adduct in rat liver by enzyme immunoassay.  

PubMed

A simple, rapid and highly sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (AFB1)-DNA adducts is reported. Polyclonal antibodies specific to the aflatoxin B1-N7-guanine adduct were produced using a novel synthetic antigen, bovine serum albumin (BSA)-guanine-AFB1. The antibodies were characterized by the Ouchterlony double diffusion technique and by antibody capture assay. The working range of the indirect competitive assay developed was between 0.45 and 330 ng of the analyte [calf thymus (CT)-DNA-AFB1]. A 50% inhibition was attained at 15 ng of the analyte (CT-DNA-AFB1). The antibody capture assay indicated that the antibody produced cross-reacted 100, 92 and 110% with BSA-guanine-AFB1, CT-DNA-AFB1 and CT-DNA-formamidopyrimidine-AFB1, respectively. When free AFB1 and guanine were used as competing analytes, the antibodies showed < or = 5% and zero cross-reactivity at the 50% inhibition level. Spiking studies indicated a recovery in the range 96-97 and 74-78% when standard CT-DNA-AFB1 was added to 10 mM phosphate buffer (pH 7.2) and control rat liver tissue, respectively. Rats exposed to a single oral dose of 1 mg kg-1 body mass of pure AFB1 were used to validate the method. The AFB1-DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB1-DNA adduct ranged between 6.06 and 7.94 micrograms mg-1 DNA. The proposed method may find application in the biological monitoring of aflatoxin B1 in molecular epidemiological studies to assess the dietary exposure of aflatoxins. PMID:9282405

Vidyasagar, T; Sujatha, N; Sashidhar, R B

1997-06-01

223

Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis.  

PubMed

An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% +/- 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% +/- 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% +/- 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% +/- 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% +/- 3.74 and CFT/ELISA was 71.72% +/- 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% +/- 4.83 and CFT/ELISA was 72.84% +/- 8.59. The Kappa index was 79.4. PMID:9100335

Vigliocco, A M; Silva Paulo, P S; Mestre, J; Briones, G C; Draghi, G; Tossi, M; Nielsen, K

1997-03-01

224

Electrochemical detection for horseradish peroxidase-based enzyme immunoassay using p-aminophenol as substrate and its application in detection of plant virus  

Microsoft Academic Search

A sensitive electrochemical method for horseradish peroxidase (HRP)-based enzyme immunoassay using p-aminophenol (PAP) as substrate is described. The enzymatic product of PAP oxidation with H2O2 catalyzed by HRP in Britton–Robinson (B–R) buffer solution of pH 4.7 is 3,4-di-[(4-hydroxyphenyl)amino]-6-[(4-hydroxyphenyl) imino]-2,4-cyclohexadiene-1-one, which yields a sensitive second order derivative linear-sweep voltammetric peak at potential of ?0.56V (versus Ag\\/AgCl) in B–R buffer solution of

Wei Sun; Kui Jiao; Shusheng Zhang; Chengliang Zhang; Zuofang Zhang

2001-01-01

225

Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay.  

PubMed Central

A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported.

Armstrong, Michael; Liu, Andrew H.; Harbeck, Ronald; Reisdorph, Rick; Rabinovitch, Nathan; Reisdorph, Nichole

2009-01-01

226

The hyper IgM syndrome  

Microsoft Academic Search

The hyper IgM syndrome is a rare, inherited immune deficiency disorder resulting from defects in the CD40 ligand\\/ CD40-signaling\\u000a pathway. X-linked hyper IgM is caused by defects in the CD40 ligand gene, while autosomal recessive hyper IgM is caused by\\u000a defects in the CD40-activated RNAediting enzyme, activation-induced cytidine deaminase, which is required for immunoglobulin\\u000a isotye switching and somatic hypermutation in

Ramsay L. Fuleihan

2001-01-01

227

A highly sensitive enzyme immunoassay for evaluation of 2'-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy.  

PubMed

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2'-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5'-succinyl-dCyd (5'sdCyd) conjugate of bovine serum albumin (5'sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5'sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5'sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of

Darwish, Ibrahim A; Mahmoud, Ashraf M; Aboul-Fadl, Tarek; Al-Majed, Abdul-Rahman A; Khalil, Nasr Y

2008-11-17

228

Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.  

PubMed

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species. PMID:23462197

Watson, Rebecca; Munro, Coralie; Edwards, Katie L; Norton, Vicki; Brown, Janine L; Walker, Susan L

2013-02-24

229

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus.  

PubMed

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient--obtained before, during, and after an infection with mumps--the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized. PMID:7453669

Nicolai-Scholten, M E; Ziegelmaier, R; Behrens, F; Höpken, W

1980-01-01

230

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus  

Microsoft Academic Search

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM.

M. E. Nicolai-Scholten; R. Ziegelmaier; F. Behrens; W. Höpken

1980-01-01

231

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor.  

PubMed

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days. PMID:10945454

Lee, W E; Thompson, H G; Hall, J G; Bader, D E

2000-01-01

232

Evaluation of a Cytomegalovirus Glycoprotein B Recombinant Enzyme Immunoassay To Discriminate between a Recent and a Past Infection  

Microsoft Academic Search

Fetal damage following cytomegalovirus (CMV) intrauterine infection is mostly linked to primary infection. To differentiate primary infection from nonprimary infection, immunoglobulin M (IgM) tests are not reliable enough, and measurement of the IgG avidity appears to be the method that is the most widely used at present. In the present study the performance of the Vidas (bioMerieux) avidity assay was

M. J. Sipewa; P. Goubau; M. Bodeus

2002-01-01

233

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

2010-01-29

234

Comparison of Performance and Cost-Effectiveness of Direct Fluorescent-Antibody, Ligase Chain Reaction, and PCR Assays for Verification of Chlamydial Enzyme Immunoassay Results for Populations with a Low to Moderate Prevalence of Chlamydia trachomatis Infection  

Microsoft Academic Search

Many laboratories use a commercial enzyme immunoassay (EIA) with verification testing to diagnose Chlamydia trachomatis infections in an effort to contain costs. This study was designed to compare the performance and cost-effectiveness of direct fluorescent-antibody assay (DFA), commercial PCR, and ligase chain reaction (LCR) for the verification of EIA results. Cervical specimens were screened by EIA. DFA, PCR, and LCR

DEBORAH DEAN; DENNIS FERRERO; MICHAEL MCCARTHY

235

Comparison of DNA Enzyme Immunoassay and Line Probe Assays (Inno-LiPA HCV I and II) for Hepatitis C Virus Genotyping  

PubMed Central

Two methods for genotyping hepatitis C virus (DNA enzyme immunoassay [DEIA] and line probe assay [Inno-LiPA HCV I and II]) were compared on 120 samples and of these 87% were assigned to the same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the core and 5? untranslated regions (UTR) were sequenced to resolve conflicts. Type 1 discordant samples had a guanosine at position ?99 in the 5? UTR, a characteristic of genotype 1b, and a core region typical of subtype 1a. The eight isolates classified as 2a/2c by LiPA and as subtype 2c by DEIA belonged to type 2.

Le Pogam, S.; Dubois, F.; Christen, R.; Raby, C.; Cavicchini, A.; Goudeau, A.

1998-01-01

236

Comparison of a high pressure liquid chromatographic analysis and an enzyme immunoassay technique for quantitation of disopyramide in serum or plasma.  

PubMed

High pressure liquid chromatographic (HPLC) and enzyme immunoassay (Emit) methods for measuring disopyramide concentrations in plasma and serum were compared. The precision of both methods was satisfactory, with all coefficients of variation in the range 1.3--6.5%. Quantitation was comparable, with a correlation coefficient of 0.991 (n = 96). There was no interference in either method from lignocaine, digoxin, propranolol, procainamide or N-monodealkylated disopyramide. HPLC was superior in terms of lower cost, the ability to quantitate metabolite concentrations, lack of interference by lipaemia or haemolysis and slightly better within-run precision. However, Emit was considered the method of choice for routine therapeutic drug monitoring because of its relative simplicity and speed of performance. PMID:7050181

Bryson, S M; Betts, A; Summer, D J; Whiting, B

1982-06-01

237

Detection of eastern equine encephalomyelitis virus and Highlands J virus antigens within mosquito pools by enzyme immunoassay (EIA). I. A laboratory study.  

PubMed

Enzyme immunoassays (EIAs) producing either chromogenic or fluorogenic end products were developed and evaluated for detection of eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses in pools of Aedes triseriatus mosquitoes. Overnight incubation of the mosquito samples in the EIA significantly enhanced the sensitivity of the test. Both the EEE and HJ EIAs were sensitive, readily detecting one infected mosquito in a pool with 99 noninfected, and specific, distinguishing homologous from the alternate alphavirus and other arboviruses. By 3 days post-infection after intrathoracic inoculation, EEE virus was isolated from 100% (30/30) of the mosquitoes examined. Concurrently, EEE virus antigen was detectable by EIA in 100% (30/30) of examined mosquitoes and by indirect fluorescent antibody (IFA) technique in 77% (23/30) of the examined mosquito head squash preparations. PMID:6091470

Hildreth, S W; Beaty, B J

1984-09-01

238

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

PubMed

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

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

1999-10-01

239

Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG), IgM, and IgA with broad specificity for enteroviruses.  

PubMed Central

An antibody-capture enzyme-linked immunosorbent assay (ELISA) with coxsackievirus B1 as the antigen was evaluated for detection of immunoglobulin G (IgG), IgM, and IgA antibodies and showed broad specificity for enteroviruses. In total, 116 serum or cerebrospinal fluid samples from 62 patients were tested by ELISA and the complement fixation test (CFT). Additionally, 15 serum samples that contained poliovirus-specific IgM antibody were tested. Serum samples from 200 healthy blood donors were used for standardization of the assays. The sensitivity of the ELISA varied with time of serum sampling, with a relatively low sensitivity when serum was collected within 3 days after the onset of symptoms (23%; 5 of 22) but good sensitivity when serum was collected later (83%; 20 of 24). The sensitivity was better than that of the CFT. The ELISAs were broadly reactive as concluded from typing of virus isolates that were simultaneously obtained. The assay did, furthermore, detect antibody against poliovirus type 3. Sera that contained rheumatoid factor, antinuclear antibody, or cardiolipin antibody (by the Venereal Disease Research Laboratory test) did not react in this ELISA. Nonspecific reactivity did occur, however, in cases of infectious mononucleosis and in Mycoplasma pneumoniae infection. The enterovirus-specific ELISA is found to be simple to perform, more sensitive than the CFT, and far less laborious than the neutralization test.

Swanink, C M; Veenstra, L; Poort, Y A; Kaan, J A; Galama, J M

1993-01-01

240

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

241

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

2008-12-14

242

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.

Harris, Michael J.; Dugger, William M.

1986-01-01

243

The IGM Project  

NASA Astrophysics Data System (ADS)

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

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

2010-01-01

244

Simultaneous Detection of Immunoglobulin A (IgA) and IgM Antibodies against Hepatitis E Virus (HEV) Is Highly Specific for Diagnosis of Acute HEV Infection  

PubMed Central

Serum samples collected from 68 patients (age, mean ± the standard deviation [SD], 56.3 ± 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 ± 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 ± 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection.

Takahashi, Masaharu; Kusakai, Shigeyuki; Mizuo, Hitoshi; Suzuki, Kazuyuki; Fujimura, Kuniko; Masuko, Kazuo; Sugai, Yoshiki; Aikawa, Tatsuya; Nishizawa, Tsutomu; Okamoto, Hiroaki

2005-01-01

245

Detection of IgG and IgM in sera from canines with blastomycosis using eight blastomyces dermatitidis yeast phase lysate antigens.  

PubMed

The objective of this study was to compare the efficacy of eight Blastomyces dermatitidis yeast phase lysate antigens (T-58: dog, Tennessee; T-27: polar bear, Tennessee; ERC-2: dog, Wisconsin; B5894: human, Minnesota; SOIL: soil, Canada; B5896: human, Minnesota; 48089: human, Zaire; 48938: bat, India) in the detection of the immunoglobulins IgG and IgM in serum specimens from canines with blastomycosis. An indirect enzyme-linked immunosorbent assay (ELISA, peroxidase system) was used to analyze sera collected during four different intervals post-infection. The yeast lysate antigen 48938 was a reactive antigen for the detection of both IgG (mean absorbance value range: 1.198-2.934) and IgM (mean absorbance value range: 0.505-0.845). For the same sera, antigen T-27 was also effective in the detection of IgG (mean absorbance value range: 0.904-3.356) and antigen 48089 was useful for the detection of IgM (mean absorbance value range: 0.377-0.554). The yeast lysate antigen B5894 proved to be a poor antigen for the detection of both IgG and IgM (mean absorbance value ranges: 0.310-0.744 for IgG, 0.025-0.069 for IgM). Inherent variations in yeast lysate antigens such as these may be utilized to develop improved immunoassay procedures for the specific detection of IgG or IgM in cases of blastomycosis. PMID:16830189

Sestero, Christine M; Scalarone, Gene M

2006-07-01

246

An indirect enzyme-linked immunoassay (ELISA) for demonstration of antibodies to Neospora caninum in serum and milk of cattle  

Microsoft Academic Search

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from cattle is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen and a mouse monoclonal antibody to bovine immunoglobulin G1 as conjugate. Western blot analysis of the iscom preparation revealed a restricted number of antigens compared with whole parasite

Camilla Björkman; O. Joakim M. Holmdahl; Arvid Uggla

1997-01-01

247

Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus  

Microsoft Academic Search

The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices.

DARCI R. SMITH; CYNTHIA A. ROSSI; TODD M. KIJEK; ERIK A. HENCHAL; GEORGE V. LUDWIG

2001-01-01

248

A comparative study of dextran coated charcoal (DCC) and enzyme immunoassay (EIA) methods for oestrogen receptor (ER) estimation in breast cancer.  

PubMed

Oestrogen receptor (ER) concentrations were measured in 100 breast cancer cytosols using both the dextran coated charcoal assay (DCC with 6--point scatchard plot) and a newly developed enzyme linked immunoassay (EIA) using a monoclonal antibody against ER. The efficiency of the two methods was compared. A highly significant correlation was observed between the ER levels determined by DCC and EIA methods (r = 0.94, p less than 0.001). The mean ER-EIA value (43.25 +/- 74.77) was, however, significantly higher than the mean ER-DCC value (18.00 + 37.27) (p less than 0.001); in both pre- menopausal (p less than 0.001) and post-menopausal (p less than 0.001) groups. Using a cut-off point at 10 fmo/mg protein for ER-EIA and 3 fmol/mg protein for ER-DCC/to distinguish ER +ve and -ve tumours, a 97% concordance between the two assays was achieved. The EIA method appears to be preferable to the DCC method because it is easy to perform, rapid, requires less tissue and does not involve the use of radioactive substances. PMID:1769684

Redkar, A A; Kabre, S S; Mittra, I

1991-03-01

249

A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings  

PubMed Central

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and ?-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaign

Jacobs, M V; Walboomers, J M M; Beek, J van; Voorhorst, F J; Verheijen, R H M; Meijer, C J L M; Brule, A J C van den; Helmerhorst, ThJ M; Snijders, P J F

1999-01-01

250

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

PubMed

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency. PMID:2645381

Verebey, K; DePace, A

1989-01-01

251

Enzyme-linked Immunoassay for Respiratory Syncytial Virus Is Not Predictive of Bronchiolitis in Sudden Infant Death Syndrome  

Microsoft Academic Search

Although respiratory syncytial virus (RSV)-infected infants may present with apnea, the role that RSV plays in sudden infant\\u000a death syndrome (SIDS) is speculative. To determine whether RSV is associated with bronchiolitis in these patients, we examined\\u000a histologic sections of lungs from 41 apparent SIDS cases and compared the results with those of enzyme-linked immunofluorescent\\u000a assay (EIA) from nasal washings. Bronchiolitis

David M. Parham; Richard Cheng; Gordon E. Schutze; Bradley Dilday; Rebecca Nelson; Stephen Erickson; Charles Kokes; Frank Peretti; William Q. Sturner

1998-01-01

252

Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis  

Microsoft Academic Search

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosor- bent assay (ELISA) and Western blot assay formats have been used to measure these

JEFFREY W. PRIEST; ANNA LI; MOHAMAD KHAN; MICHAEL J. ARROWOOD; PATRICK J. LAMMIE; CORINNE S. ONG; JACQUELIN M. ROBERTS; JUDITH ISAAC-RENTON

2001-01-01

253

Magnetic affinity enzyme-linked immunoassay based on recombinant 26 kDa glutathione-S-transferase for serological diagnosis of schistosomiasis japonica.  

PubMed

Schistosomiasis remains a serious worldwide public health problem. Improving the diagnostic assay for surveillance and monitoring will contribute to hastening the possible elimination of the disease in endemic regions. Therefore, this study aims to develop magnetic affinity enzyme-linked immunoassay (MEIA) for serological diagnosis of schistosomiasis based on recombinant 26kDa glutathione-S-transferase of Schistosoma japonicum (rSj26GST). BALB/c mice infected with S. japonicum cercariae (40 per mouse) were used. After infecting for 6 weeks, the antibody was detected by MEIA. All of the infected mouse sera were effectively determined by MEIA. Compared with the enzyme-linked immunosorbent assay (ELISA), MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio (3.92 versus 2.66). MEIA was further applied for diagnosis of human schistosomiasis. Sera from 28 schistosomiasis-confirmed patients with low-intensity infection, 15 treated patients, and 20 non-endemic negative controls, were used to assess the assay. The results showed that MEIA and ELISA had similarity in positive detection rates. However, the higher P/N of MEIA was observed at the same dilution ratio. MEIA had high negative rate in detection of specific IgG in the treated patients. Moreover, there was no cross reaction with the sera of paragonimiasis patients. These results suggested that MEIA based on rSj26GST is a simple, rapid, convenient assay for the diagnosis of schistosomiasis. PMID:22940100

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

2012-08-20

254

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

255

Detection of Helicobacter pylori antibodies in a pediatric population: comparison of three commercially available serological tests and one in-house enzyme immunoassay.  

PubMed

A serum immunoglobulin G enzyme immunoassay (EIA) for Helicobacter pylori antibodies already in use in adults was evaluated with 99 pediatric serum samples to determine its usefulness for the study of H. pylori disease in children. The reference method used was either the (13)C-urea breath test or a biopsy culture of gastric mucosa. In children, an EIA cutoff of 0.35 absorbancy unit yielded sensitivity, specificity, and positive and negative predictive values of 93, 97, 93, and 97%, respectively. The cutoff recommended when this EIA was published for use in adults was 0.70 absorbancy unit (H. Gnarpe, P. Unge, C. Blomqvist, and S. Mäkitalo, APMIS 96:128-132, 1988). Another subset of 169 serum samples taken from children was analyzed by four serological tests in order to compare the performance of the in-house EIA with the Pyloriset, HM-CAP, and Helico-G kits. For the 169 samples, 10 (5.9%) false-positives and no false-negatives occurred with the Helico-G, 3 (1.8%) false-positives and no false-negatives occurred with the Pyloriset, and 3 (1.8%) false-positives and 1 (0.6%) false-negative occurred with the HM-CAP. For the 169 samples, 1 (0.6%) false-positive and no false-negatives occurred with the in-house EIA. Serological detection of H. pylori antibodies with our EIA seems to be valuable in diagnosing H. pylori infection in children, but only if a lowered, specific pediatric cutoff is established. The commercial kits, particularly the Helico-G, seem to overdiagnose pediatric H. pylori infection. A positive serological test for H. pylori infection, particularly for children, needs to be confirmed by a reference method because of the possibility of spontaneous eradication of infection, with a lingering serological response. PMID:10488200

Sunnerstam, B; Kjerstadius, T; Jansson, L; Giesecke, J; Bergström, M; Ejderhamn, J

1999-10-01

256

Prospective comparison of a commercial multiplex real-time polymerase chain reaction and an enzyme immunoassay with toxigenic culture in the diagnosis of Clostridium difficile-associated infections.  

PubMed

Clostridium difficile infections (CDI) is a leading cause of nosocomial infections worldwide. The changes in the epidemiology of CDI during the past years, including the appearance of new epidemic strains of C. difficile that cause CDI episodes with increased severity, have led to the development of molecular methods with improved sensitivity and specificity. This study was designed to compare the performances of one antigen assay (Vidas, bioMérieux) and one molecular assay (GeneXpert, Cepheid). Fecal specimens from hospitalized patients (n = 230) suspected of having CDI were tested by both assays. Eleven specimens were positive and 202 were negative for both methods. After discrepant analysis by C. difficile toxigenic culture with broth enrichment and neutralization assay, the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 23 (10%) and 206 (89.6%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value for GeneXpert were 91.7%, 99%, 91.7%, and 99%, and for Vidas were 48%, 99%, 84.6%, and 94.5%, respectively. The sensitivity and PPV of polymerase chain reactoin GeneXpert assay far exceeded those of the EIA Vidas assay. The clinical characteristics of concordant and discrepant study patients were similar with the exception of the number of previous CDI episodes, which were higher in the concordant study patients; the clinical characteristics of both groups were similar. In conclusion, due to the appearance of more virulent strains of C. difficile during the last years that have produced dramatic changes in the epidemiology of C. difficile, we recommend that toxin enzyme immunoassays be replaced with rapid molecular-based tests for toxigenic C. difficile. PMID:23415540

Hernández-Rocha, Cristian; Barra-Carrasco, Jonathan; Álvarez-Lobos, Manuel; Paredes-Sabja, Daniel; Guzmán-Durán, Ana María

2013-02-12

257

Development and validation of a simple, sensitive, second antibody format enzyme immunoassay (EIA) for LH determination in mithun (Bos frontalis) plasma.  

PubMed

The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin-streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 microL mithun plasma. The LH standards ranging from 6.25 pg/well/20 microL to 400 pg/well/20 microL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 microL. Plasma volumes for the EIA viz. 10 and 20 microL did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 microg i.v., with a synthetic analogue of GnRH (Buserelin-Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun. PMID:15794124

Mondal, Mohan; Dhali, Arindam; Prakash, Bhukya; Rajkhowa, Chandan; Prakash, B S

2005-01-01

258

Highly sensitive second-antibody enzyme immunoassay for determination of estradiol-17beta concentration in blood plasma of the mithun (Bos frontalis).  

PubMed

The objective of the present study was to develop and validate a simple, sensitive, quick and economic enzyme immunoassay (EIA) for estradiol-17beta (E2) in mithun (Bos frontalis) plasma on microtiter plates using a second-antibody coating technique and hormone-horseradish peroxidase as a label. For the assay, the wells of microtiter plates were coated with affinity-purified goat anti-rabbit IgG that binds the hormone-specific antibody. One milliliter of mithun plasma was extracted using benzene and 50 microl of 300 microl volume reconstituted with assay buffer was run in the assay along with standards ranging from 0.10-100 pg/well prepared in assay buffer. The sensitivity of the assay was 0.72 pg/ml. The intra- and inter-assay coefficients of variation were below 10%, and the extraction efficiency was >93%. Linearity of recovery of the added hormone concentrations was recorded. The assay developed was further validated biologically by estimating the hormone concentrations in six female and five male mithun calves, 12 cyclic mithuns for the entire reproductive cycle, and four pregnant mithun cows. The EIA developed can estimate low concentrations of E2 (2.2-5.2 pg/ml) in growing calves as well as very high concentrations of the hormone during pregnancy (E2=85.6-143.5 pg/ml). Apart from being non-radioactive, the assay developed has several advantages over conventional radioimmunoassays: it is more sensitive, less labor intensive, simpler to perform, and less time consuming. In conclusion, the EIA procedure described herein is sufficiently reliable, economic, safe, quick and sensitive to estimate the hormone at all physiological levels in bovine plasma. PMID:17867839

Mondal, Mohan; Prakash, Bukkaraya Samudram

2007-04-01

259

Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk.  

PubMed

The objective of this study was to compare 2 enzyme immunoassays (EIAs) with a radioimmunoassay (RIA) as to sensitivity and accuracy in the measurement of the progesterone (P4) concentration in bovine plasma, skim milk, and whole milk. The 72 samples from 24 lactating dairy cows expected to have either a high P4 concentration (cows in diestrus or pregnant) or a low P4 concentration (cows in estrus or anestrus) were analyzed by RIA, solid-phase EIA (SPEIA), which included a solvent extraction step, or direct EIA (DEIA) without solvent extraction. The overall mean concentrations of P4 did not differ (P < 0.4) among the assays. However, for the cows that were in diestrus or pregnant, the mean P4 concentrations (and standard error) were higher (P < 0.03), regardless of sample type, with RIA than with SPEIA, at 7.3 (0.7) and 6.1 (0.6) ng/mL, respectively. When only the high-P4 samples analyzed by RIA were compared, the mean P4 concentration was higher (P < 0.001) in whole milk than in skim milk, at 9.8 (1.0) and 4.1 (0.7) ng/mL, respectively. Although the mean P4 concentrations in the low-P4 samples did not differ (P < 0.80) among assays, the proportions of cows with a P4 concentration > or = 1 ng/mL were 3%, 14%, and 44% for RIA, SPEIA, and DEIA, respectively (P < 0.01; DEIA > SPEIA > RIA). PMID:18214159

Colazo, Marcos G; Ambrose, Divakar J; Kastelic, John P; Small, Julie A

2008-01-01

260

Correlation between Clostridium difficile Bacterial Load, Commercial Real-Time PCR Cycle Thresholds, and Results of Diagnostic Tests Based on Enzyme Immunoassay and Cell Culture Cytotoxicity Assay.  

PubMed

The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (CT) with the results of quantitative culture using Spearman's rank correlation coefficient. Finally, we sequenced the genomes of 24 strains with different detection profiles. A total of 203 clinical samples harboring toxigenic C. difficile were analyzed and sorted into one of four groups: 17 PCR(+) (group 1), 37 PCR(+) GDH(+) (group 2), 24 PCR(+) GDH(+) CCA(+) (group 3), and 125 PCR(+) GDH(+) ToxAB(+) (group 4). The overall median fecal load in log10 CFU/g was 6.67 (interquartile range [IQR], 5.57 to 7.54). The median fecal bacterial load of groups 1, 2, 3, and 4 were 4.15 (IQR, 3.00 to 4.98), 5.74 (IQR, 4.75 to 6.16), 6.20 (IQR, 5.23 to 6.80), and 7.08 (IQR, 6.35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR CT and fecal loads (? = -0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P = 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests. PMID:23966497

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

2013-08-21

261

Large-Scale Evaluation of the Immuno-Mycologics Lateral Flow and Enzyme-Linked Immunoassays for Detection of Cryptococcal Antigen in Serum and Cerebrospinal Fluid  

PubMed Central

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

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

2013-01-01

262

Identification of Performance Problems in a Commercial Human Immunodeficiency Virus Type 1 Enzyme Immunoassay by Multiuser External Quality Control Monitoring and Real-Time Data Analysis? †  

PubMed Central

In June 2005, a pilot program was implemented in Canadian laboratories to monitor the performance of the Abbott human immunodeficiency virus types 1 and 2 (HIV-1/2) gO enzyme immunoassay (EIA). Two different external quality control (QC) reagents and a “real-time” software analysis program were evaluated. In November 2005, higher-than-expected calibrator rate values in these kits were first reported at the Ontario Ministry of Health (Etobicoke), followed by the Alberta Provincial Public Health Laboratory (Edmonton and Calgary) and others. These aberrations were easily and readily tracked in “real time” using the external QC reagents and the software program. These high calibrator values were confirmed in Delkenheim, Germany, by Abbott, and a manufacturing change was initiated beginning with lot 38299LU00, which was distributed to laboratories in Canada in April 2006. However, widespread reports of calibrator failure by laboratories outside Canada were made in March 2006. In April 2006, Abbott Diagnostics initiated a level III investigation to identify the root cause, which was prolonged storage, under uncontrolled storage conditions, of the raw material used in the manufacture of the matrix cells. To the best of our knowledge, this is the first example of a program in Canada for serological testing that combines a common external QC reagent and a “real-time” software program to allow laboratories to monitor kit performance. In this case, external QC monitoring helped identify and confirm performance problems in the Abbott HIV-1/2 gO EIA kit, further highlighting the benefit of implementing such a program in a national or multilaboratory setting for laboratories performing diagnostic and clinical monitoring testing.

Kim, J.; Swantee, C.; Lee, B.; Gunning, H.; Chow, A.; Sidaway, F.; Sherlock, C.; Garceau, R.; Dimech, W.; Malloch, L.

2009-01-01

263

Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer  

PubMed Central

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

Maine, G. T.; Stricker, R.; Schuler, M.; Spesard, J.; Brojanac, S.; Iriarte, B.; Herwig, K.; Gramins, T.; Combs, B.; Wise, J.; Simmons, H.; Gram, T.; Lonze, J.; Ruzicki, D.; Byrne, B.; Clifton, J. D.; Chovan, L. E.; Wachta, D.; Holas, C.; Wang, D.; Wilson, T.; Tomazic-Allen, S.; Clements, M. A.; Wright, G. L.; Lazzarotto, T.; Ripalti, A.; Landini, M. P.

2000-01-01

264

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

PubMed

We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring. PMID:23946517

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

2013-08-14

265

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

PubMed

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P < 0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hormone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used in RIA procedures. PMID:16908963

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

2006-07-01

266

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

267

A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG.  

PubMed

An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102). PMID:3905782

Kohno, T; Hashida, S; Ishikawa, E

1985-08-01

268

Development of an enzyme-linked immunoassay for the quantification of YKL-40 (cartilage gp-39) in guinea pig serum using hen egg yolk antibodies  

Microsoft Academic Search

An indirect competition immunoassay for the quantification of YKL-40 (cartilage gp-39, Chondrex) in guinea pig serum has been developed using egg yolk antibodies (IgY). The immune response of hens to YKL-40 was verified by immunoblot analyses. Highly specific antibodies were obtained 30 days after the first injection. The ELISA was developed in 96-well microtiter plates with quadruplicate determinations for each

Frédéric De Ceuninck; Philippe Pastoureau; Séverine Agnellet; Jacqueline Bonnet; Paul Michel Vanhoutte

2001-01-01

269

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

Microsoft Academic Search

A zirconia (ZrO2) 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 ZrO2 film with uniform

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

2009-01-01

270

Detection of Staphylococcus aureus cell walls by enzyme-linked immunoassay using antibodies prepared from a semi-synthetic peptidoglycan precursor.  

PubMed

The peptidoglycan layer of Staphylococcus aureus contains a (Gly)(5) cross-link which is not found in other bacteria, and which could be used to develop a specific immunoassay for detection of S. aureus in MRSA infections. A semi-synthetic route was used to prepare the S. aureus peptidoglycan precursor UDPMurNAc-L-Ala-?-D-Glu-L-Lys(Gly)(5)-D-Ala-D-Ala, which was covalently attached to carrier protein bovine serum albumin via the UDP nucleotide. Serum raised using this antigen showed specificity for chemically immobilised peptidoglycan monomer containing (Gly)(5), using an ELISA immunoassay. ELISA assays using 0.1 or 1.0 ?g samples of cell walls prepared from two MRSA strains and one penicillin-sensitive S. aureus strain, and from three other bacteria, showed the highest response against cell walls containing (Gly)(5), with a particularly high response against cell walls from one MRSA strain. Competition assays to investigate antibody selectivity demonstrated that the antibody response could be most effectively antagonised using ligands containing (Gly)(5). These data demonstrate that it is possible to generate antibodies with high affinity and selectivity for the (Gly)(5) containing monomer in S. aureus peptidoglycan, that could be used to develop an immunoassay for S. aureus. PMID:22249441

Sandhu, Sandeep; Schouten, James A; Thompson, Julie; Davis, Mark; Bugg, Timothy D H

2012-01-17

271

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

PubMed Central

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

Arlestig, Lisbeth; Mullazehi, Mohammed; Kokkonen, Heidi; Rocklov, Joacim; Ronnelid, Johan; Dahlqvist, Solbritt Rantapaa

2012-01-01

272

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

273

Deficiency of IgM.  

National Technical Information Service (NTIS)

An 8 1/2-month-old infant with absent IgM had recurrent Pseudomonas infections. IgG and IgA, but no IgM-containing plasma cells, were identified in the spleen by immunofluorescence. The spleen and lymph nodes lacked germinal centers, but Peyer's patches a...

W. P. Faulk W. S. Kiyasu M. D. Cooper H. H. Fudenberg

1970-01-01

274

Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein  

PubMed Central

Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR?/? mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Horkko, Sohvi

2012-01-01

275

Laboratory diagnosis of congenital syphilis by immunoglobulin M (IgM) and IgA immunoblotting.  

PubMed Central

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants. Images

Schmitz, J L; Gertis, K S; Mauney, C; Stamm, L V; Folds, J D

1994-01-01

276

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...

277

Electrochemiluminescence flow injection immunoassay for atrazine  

Microsoft Academic Search

Antibodies to atrazine were labelled with glucose oxidase and used in colorimetric enzyme linked immunosorbent assays. Transparent aminosilanized indium tin oxide coated glass electrodes were derivatized with aminodextran covalently modified with atrazine caproic acid. The labelled antibodies were used to investigate the derivatized electrodes colorimetrically and the electrodes were use in an electrochemiluminescence flow injection analyser. Electrochemiluminescence immunoassay for atrazine

Robert Wilson; Michael H. Barker; David J. Schiffrin; Ram Abuknesha

1997-01-01

278

Immunoassays in Biotechnology  

EPA Science Inventory

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

279

Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens  

PubMed Central

BACKGROUND—The increasing use of macrolides especially in the treatment of Helicobacter pylori infection has led to an increase in resistant strains. The resistance of H pylori to macrolides, especially clarithromycin, is one of the major causes of eradication failure. In H pylori, clarithromycin resistance is due to point mutations localised in domain V of 23S rRNA. ?AIM—To develop a molecular technique based on amplification of a relevant fragment of the 23S rRNA and colorimetric hybridisation in liquid phase to detect directly in biopsy specimens the type of mutation associated with resistance of H pylori to clarithromycin. ?METHODS—Gastric biopsy samples from 61 patients were submitted to this test. The results were compared with standard methods (determination of minimal inhibition concentration, polymerase chain reaction/restriction fragment length polymorphism, and/or DNA sequencing) in order to evaluate the test and to define the cut off values, specificity, and sensitivity. ?RESULTS—The 14 biopsy samples in which H pylori was not detected did not give a positive result in any assay, and the 14 samples harbouring strains susceptible to clarithromycin gave a positive result with the wild type probe as expected. The 33 biopsy specimens containing resistant strains always gave a positive signal with one of the probes detecting resistant organisms, but in eight cases they also reacted with the wild type probe, indicating that a mixture of resistant and susceptible organisms was present. ?CONCLUSION—The importance of this new assay is that it allows the detection of multiple genotypes corresponding to either heterogeneous genotypes or mixed infections. Moreover, it allows in a single step not only the detection of H pylori but also the determination of its susceptibility to clarithromycin directly in biopsy specimens without the need for culture. ?? Keywords: Helicobacter pylori; resistance; clarithromycin; macrolide; polymerase chain reaction (PCR); immunoassay

Marais, A; Monteiro, L; Occhialini, A; Pina, M; Lamouliatte, H; Megraud, F

1999-01-01

280

Increased concentration of two different advanced glycation end-products detected by enzyme immunoassays with new monoclonal antibodies in sera of patients with rheumatoid arthritis  

PubMed Central

Background Levels of pentosidine (representative of advanced glycation end-products) in sera of patients with rheumatoid arthritis are increased when compared with sera of other diagnoses or healthy controls. These levels have been reported to correlate with clinical indices of rheumatoid arthritis activity and with laboratory markers of inflammation. The purpose of this study was to find out if these findings pertain to other advanced glycation end-products. Methods We have developed two immunoassays based on new monoclonal antibodies to advanced glycation end-products. Antibody 103-E3 reacts with an unidentified antigen, formed in the reaction of proteins with ribose, while antibody 8-C1 responds to N?-(carboxyethyl)lysine. We have used these monoclonal antibodies to measure levels of advanced glycation end-products in sera of patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and healthy controls. We calculated the correlations between advanced glycation end-product levels in rheumatoid arthritis sera and the Disease Activity Score 28 (DAS28), age, disease duration, CRP, anti-CCP, rheumatoid factor and treatment with corticosteroids, respectively. Results Levels of both glycation products were significantly higher in sera of patients with rheumatoid arthritis when compared with sera of patients with systemic lupus erythematosus, osteoarthritis, or the healthy controls. Neither the level of N?-(carboxyethyl)lysine nor the level of the 103-E3 antigen in rheumatoid arthritis sera correlated with the DAS28-scored rheumatoid arthritis activity. The levels of both antigens in rheumatoid arthritis sera did not correlate with age, gender, corticosteroid treatment, or levels of CRP, anti-CCP antibodies, and rheumatoid factor in sera. Conclusions We report highly specific increases in the levels of two advanced glycation end-products in sera of patients with rheumatoid arthritis. This increase could be explained neither by rheumatoid arthritis activity nor by inflammation. We propose a working hypothesis that presumes the existence of a link between advanced glycation end-product formation and induction of autoimmunity.

2010-01-01

281

Performance of two commercially available automated immunoassays for the determination of Epstein-Barr virus serological status.  

PubMed

This study evaluated the performance of two automated Vidas (V) and Liaison (L) immunoassays for Epstein-Barr virus (EBV) serology. The detection of the viral capsid antigen (VCA) IgM, the VCA/early antigen (VCA/EA) IgG, and the Epstein-Barr nuclear antigen (EBNA) IgG was assessed on 526 sera collected for routine EBV testing in immunocompetent subjects. The determination of expected EBV status (186 EBV primary infections, 183 past EBV infections, and 157 EBV-seronegative individuals) was based on results of routine laboratory enzyme immunoassays (EIAs) together with clinical data. The sensitivity and specificity of each individual marker were determined in comparison to the expected EBV status. The agreement between the V and L profiles and the expected EBV status was established through the interpretation of combinations of the different EBV markers. Statistically significant differences between the two tests were found for the specificity of the VCA IgM marker (96.2% for V versus 93.2% for L), the sensitivity of the VCA/EA IgG marker (89% for V versus 94% for L), and the specificity of the EBNA IgG marker (96.5% for V versus 74.2% for L). The results determined for the two assays with respect to overall agreement with the established expected EBV status were not significantly different (89.7% for V versus 88.2% for L), with discrepancies mainly observed in sera referenced as primary infections. These findings demonstrated the similar performances of the Vidas and the Liaison assays for the establishment of an EBV serological status using the VCA, EA, and EBNA markers. PMID:22539474

Lupo, J; Germi, R; Semenova, T; Buisson, M; Seigneurin, J M; Morand, P

2012-04-25

282

Performance of Two Commercially Available Automated Immunoassays for the Determination of Epstein-Barr Virus Serological Status  

PubMed Central

This study evaluated the performance of two automated Vidas (V) and Liaison (L) immunoassays for Epstein-Barr virus (EBV) serology. The detection of the viral capsid antigen (VCA) IgM, the VCA/early antigen (VCA/EA) IgG, and the Epstein-Barr nuclear antigen (EBNA) IgG was assessed on 526 sera collected for routine EBV testing in immunocompetent subjects. The determination of expected EBV status (186 EBV primary infections, 183 past EBV infections, and 157 EBV-seronegative individuals) was based on results of routine laboratory enzyme immunoassays (EIAs) together with clinical data. The sensitivity and specificity of each individual marker were determined in comparison to the expected EBV status. The agreement between the V and L profiles and the expected EBV status was established through the interpretation of combinations of the different EBV markers. Statistically significant differences between the two tests were found for the specificity of the VCA IgM marker (96.2% for V versus 93.2% for L), the sensitivity of the VCA/EA IgG marker (89% for V versus 94% for L), and the specificity of the EBNA IgG marker (96.5% for V versus 74.2% for L). The results determined for the two assays with respect to overall agreement with the established expected EBV status were not significantly different (89.7% for V versus 88.2% for L), with discrepancies mainly observed in sera referenced as primary infections. These findings demonstrated the similar performances of the Vidas and the Liaison assays for the establishment of an EBV serological status using the VCA, EA, and EBNA markers.

Lupo, J.; Germi, R.; Semenova, T.; Buisson, M.; Seigneurin, J. M.

2012-01-01

283

Detection of Giardia lamblia, Entamoeba histolytica\\/Entamoeba dispar, and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay  

Microsoft Academic Search

The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immuno- assay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica\\/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with

LYNNE S. GARCIA; ROBYN Y. SHIMIZU; CAROLINE N. BERNARD

2000-01-01

284

Comparison of an enzyme immunoassay and a latex agglutination system for the diagnosis of invasive aspergillosis in bone marrow transplant recipients  

Microsoft Academic Search

The performance of two Aspergillus antigenemia systems, the sandwich enzyme-linked immunosorbent assay (ELISA), Platelia Aspergillus test, and the latex agglutination (LA), Pastorex Aspergillus test, in the diagnosis of invasive aspergillosis were compared by testing 364 serum samples from 22 bone marrow transplant (BMT) recipients. Sensitivity and specificity for the ELISA test were 60% and 82% respectively, vs 40% and 94%

M Machetti; M Feasi; N Mordini; MT Van Lint; A Bacigalupo; JP Latgé; J Sarfati; C Viscoli

1998-01-01

285

A Quantitative Nitrocellulose Enzyme Immunoassay and its Application to the Screening of Hybrbidomas for the Detection of Uncharacterized Tumor-Associated Antigens in Sera of Cancer Patients  

Microsoft Academic Search

An enzyme-linked immunosorbent assay on nitrocellulose based microtiter plates for the detection of uncharacterized tumor associated antigens in squamous cell carcinoma and adenocarcinoma cancer patients' sera is described. Nitrocellulose microtiter plates are more sensitive than the plastic plates of polystyrene and polyvinyl chloride for the detection of antigens in serum. Monoclonal antibodies were selected for their net reactivities toward cancer

Y. S. Victor Liu; Janice K. Daniels; Patricia A. Nelson

1988-01-01

286

Microfluidic Chips for Immunoassays  

NASA Astrophysics Data System (ADS)

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.

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

2013-06-01

287

Mutations in Activation-Induced Cytidine Deaminase in Patients with Hyper IgM Syndrome  

Microsoft Academic Search

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common

Yoshiyuki Minegishi; Aubert Lavoie; Charlotte Cunningham-Rundles; Pierre-Michel Bédard; Jacques Hébert; Louise Côté; Kazuo Dan; Debra Sedlak; Rebecca H. Buckley; Alain Fischer; Anne Durandy; Mary Ellen Conley

2000-01-01

288

Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2' using a bioluminescent enzyme immunoassay.  

PubMed

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2' (PBP2') using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50 pg/ml and recombinant PBP2' at 500 pg/ml. PBP2 and PBP2' present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2' extracted from 10(6) colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2' positive. The 34 MSSA specimens were PBP2 positive and PBP2' negative. Moreover, the BLEIA could detect PBP2' extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2' may be useful in clinical diagnostics. PMID:23220099

Shiga, Kazuki; Gomi, Keiko; Nishimura, Motoi; Watanabe, Masaharu; Nomura, Fumio; Kajiyama, Naoki

2012-12-06

289

A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings.  

PubMed Central

Two cocktails of digoxigenin-labeled human papillomavirus (HPV) type-specific oligonucleotide probes and an enzyme immunoassay (EIA) were used as a basis to developed a group-specific detection method for 14 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk (types 6, 11, 40, 42, 43, and 44) HPVs, following a general primer GP5+/bioGP6(+)-mediated PCR. The sensitivity of this high-risk/low-risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type. Comparison of HR/LR HPV PCR-EIA with radioactive Southern blot hybridization using a general probe on the same PCR products derived from 417 cytomorphologically abnormal cervical scrapings resulted in an overall agreement of 96% between the two methods. Complete concordance between group-specific HR/LR detection and individual typing results for both single and multiple infections indicate the strong specificity of this HR/LR HPV PCR-EIA. Multiple infections could be predicted by comparing PCR-EIA optical density values of the cocktail probes with one of the individual oligonucleotide probes. This novel HR/LR PCR-EIA allows accurate and rapid identification of high-risk and low-risk HPV types in cervical scrapings and will facilitate HPV detection in HPV mass-screening programs.

Jacobs, M V; Snijders, P J; van den Brule, A J; Helmerhorst, T J; Meijer, C J; Walboomers, J M

1997-01-01

290

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.

McGowan, Karin L.

2012-01-01

291

Updates in immunoassays: parasitology.  

PubMed

Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal. PMID:22953520

Josko, Deborah

2012-01-01

292

BrucELISA: an enzyme-antibody immunoassay for detection of Brucella abortus antibodies in milk: correlation with the Brucella ring test and with shedding of viable organisms.  

PubMed Central

An indirect enzyme-antibody immunosorbent assay (BrucELISA) is described for the detection of antibody to Brucella abortus in cow's milk. Three series of milk samples were obtained from an adult-vaccinated dairy herd infected with B. abortus. The BrucELISA system was used as a screening test for individual milks diluted 1:200 (BE 200 test), for undiluted bulk milks, and to determine antibody titer (BrucELISA titration assay). The BrucELISA results correlated highly with positive Brucella ring test reactions and culture positivity, eliminated false-positive Brucella ring test reactions, detected antibody in some samples which were Brucella ring test negative, and distinguished between vaccinated and infected animals. BrucELISA titration assay titers of greater than 1:800 were correlated with shedding, or were prognostic for animals which eventually became shedders. Binding of the enzyme-antibody conjugate to bovine immunoglobulin in the absence of rabbit anti-bovine immunoglobulin occurred with culture-positive or -negative milks showing titers of greater than 1:1,600 (the beta effect); the effect was also of predictive value in identifying eventual shedders. The BrucELISA system is a sensitive, specific, and inexpensive method for screening large numbers of individual or bulk milk samples for the presence of antibody to B. abortus.

Boraker, D K; Stinebring, W R; Kunkel, J R

1981-01-01

293

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

294

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test.  

PubMed

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo-encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 degrees C. CSF end-titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme-linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut-off value of >or= 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end-titres >or= 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end-titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine. PMID:12167095

Lejon, V; Legros, D; Richer, M; Ruiz, J A; Jamonneau, V; Truc, P; Doua, F; Djé, N; N'Siesi, F X; Bisser, S; Magnus, E; Wouters, I; Konings, J; Vervoort, T; Sultan, F; Büscher, P

2002-08-01

295

Immunoassay of infectious agents.  

PubMed

Immunoassays have evolved for a broad range of applications since the pioneering work of Yalow and Berson who developed the first competitive radioimmunoassay (RIA) for human insulin in 1959. Immunoassay detection of specific antigens and host-produced antibodies directed against such antigens consitutes one of the most widely used and successful methods for diagnosing infectious diseases (IDs). The number and variety of new assay systems that are continually being developed reflect the increasing demand for immunoassays possessing greater sensitivity, speed, and ease of use. This trend has been driven, in part, by the need for improved immunodiagnostic systems to perform rapid testing and counter emerging IDs and biothreat (BT) agents. Another factor driving this trend is the need to integrate immunoassays with more sensitive nucleic acid-based methods for a comprehensive approach. Here we examine the development of immunoassays, some of the key formats used for the detection and identification of BT/ID agents, and the application of these technologies under different scenarios. PMID:14579751

Andreotti, Peter E; Ludwig, George V; Peruski, Anne Harwood; Tuite, James J; Morse, Stephen S; Peruski, Leonard F

2003-10-01

296

System for Gel Electrophoretic Immunoassay.  

National Technical Information Service (NTIS)

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic s...

A. E. Herr A. K. Singh D. J. Throckmorton

2005-01-01

297

Comparison of the Syva MicroTrak enzyme immunoassay and Gen-Probe PACE 2 with cell culture for diagnosis of cervical Chlamydia trachomatis infection in a high-prevalence female population.  

PubMed Central

Culture is currently considered the "gold standard" for detecting Chlamydia trachomatis infections. We evaluated the Syva MicroTrak enzyme immunoassay (EIA) and Gen-Probe PACE 2 tests, which detect chlamydial antigens and rRNA, respectively. These assays were compared with each other and with culture for the detection of C. trachomatis in cervical specimens obtained from 217 women attending a clinic for sexually transmitted diseases. The prevalence of infection was 22.1% by culture. The sensitivity, specificity, and positive and negative predictive values were 79.2, 98.2, 92.6, and 94.3%, respectively, for EIA. For PACE 2, the respective values were 77.1, 97.6, 90.1, and 93.7%. After corrections for two false-negative cultures, the sensitivities and specificities were 80 and 99.4%, respectively, for the EIA and 78 and 98.8%, respectively, for the probe assay. Quantitative evaluation of the results showed that false-negative results with either assay were associated with cultures that had low inclusion counts or were negative without subpassage. Analysis of nonculture results revealed that 2.3% of the EIA results and 4.6% of the probe assay results were within +/- 30% of the respective assay cutoff values. These included four false-negative (one EIA and three probe) and two false-positive (one EIA and one probe) results. The Syva MicroTrak EIA and the Gen-Probe PACE 2 assay are comparable to but significantly less sensitive than culture. Use of a grey zone may help identify the need for repeat or confirmatory testing.

Clarke, L M; Sierra, M F; Daidone, B J; Lopez, N; Covino, J M; McCormack, W M

1993-01-01

298

Long-Term Follow-Up of Hepatitis B Surface Antibody Levels in Subjects Receiving Universal Hepatitis B Vaccination in Infancy in an Area of Hyperendemicity: Correlation between Radioimmunoassay and Enzyme Immunoassay  

PubMed Central

The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In nonboosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r = 0.91; P < 0.0001). An equation for RIA to EIA level conversion was established: log EIA titer = ?0.12 + (1.31 · log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hyporesponders or individuals at risk in adolescence.

Wang, Ching-Wen; Wang, Li-Chieh; Chang, Mei-Hwei; Ni, Yen-Hsuan; Chen, Huey-Ling; Hsu, Hong-Yuan; Chen, Ding-Shin

2005-01-01

299

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

PubMed

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

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

300

Comparison of Performance and Cost-Effectiveness of Direct Fluorescent-Antibody, Ligase Chain Reaction, and PCR Assays for Verification of Chlamydial Enzyme Immunoassay Results for Populations with a Low to Moderate Prevalence of Chlamydia trachomatis Infection  

PubMed Central

Many laboratories use a commercial enzyme immunoassay (EIA) with verification testing to diagnose Chlamydia trachomatis infections in an effort to contain costs. This study was designed to compare the performance and cost-effectiveness of direct fluorescent-antibody assay (DFA), commercial PCR, and ligase chain reaction (LCR) for the verification of EIA results. Cervical specimens were screened by EIA. DFA, PCR, and LCR were compared as verification tests for EIA-reactive specimens and negative greyzone (NGZ) specimens at 50% below the cutoff value. These samples were also tested by in-house PCR, which was used in the analysis of verification results. A total of 477 (7%) of 6,571 samples were reactive or within the NGZ. EIA results with verification by DFA testing (EIA/DFA results) agreed with 93% of the true results compared with 97% for EIA/PCR results for one set of 242 samples; there was 97% agreement with true results for EIA/DFA results versus 95% for EIA/LCR results for another set of 235 samples. Ten samples were false positive by LCR. Time and costs were equivalent for EIA with the DFA, PCR, or LCR as the verification test but were two- to threefold greater for PCR or LCR alone than for EIA with verification. Since it is important to balance cost containment with public health objectives, DFA, PCR, and LCR as EIA verification tests for cervical samples offer acceptable sensitivities and specificities at reasonable cost for low- to moderate-risk populations and therefore can be extended to a broader spectrum of at-risk populations.

Dean, Deborah; Ferrero, Dennis; McCarthy, Michael

1998-01-01

301

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.

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

2013-01-01

302

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

303

Isotope-labeled immunoassays without radiation waste  

PubMed Central

The practice of immunoassay has experienced a widespread transition from radioisotopic labeling to nonisotopic labeling over the last two decades. Radioisotope labels have drawbacks that hamper their applications: (i) perceived radiation hazards of reagents, (ii) regulatory requirements and disposal problems of working with radioactive materials, and (iii) short shelf-life of the labeled reagents. The advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity, as is often the case with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay with the long-life isotope 14C as the label and accelerator mass spectrometer (AMS) as the detection system. AMS quantifies attomole levels of several isotopes, including 14C. With this exquisite sensitivity, the sensitivity of an immunoassay is limited by the Kd of the antibody and not the detection system. The detection limit of the assays for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin was 2.0 × 10?10 M and 2.0 × 10?11 M, respectively, approximately an order of magnitude below the standard enzyme immunoassay. Notably, <1 dpm (0.45 pCi) of 14C-labeled compound was used in each assay, which is well below the limit of disposal (50 nCi per g) as nonradioactive waste. Thus, endogenous reporter ligands quantified by AMS provide the advantages of an RIA without the associated problems of radioactive waste.

Shan, Guomin; Huang, Wei; Gee, Shirley J.; Buchholz, Bruce A.; Vogel, John S.; Hammock, Bruce D.

2000-01-01

304

Combined Determination of Coxiella burnetii-Specific Immunoglobulin M (IgM) and IgA Improves Specificity in the Diagnosis of Acute Q Fever  

Microsoft Academic Search

Immunoglobulin M (IgM) and IgA responses in patients with acute Q fever were compared by enzyme-linked immunosorbent assay. An increase in both IgM and IgA was observed in paired sera from all 19 patients with acute Q fever, and both IgM and IgA levels showed good correlation with complement fixation test titers. Paired sera from 23 patients with infections other

PETER DEVINE; CATHERINE DOYLE; GEOFF LAMBKIN

1997-01-01

305

IgM and IgG Antibodies to Phenolic Glycolipid I from Mycobacterium leprae in Leprosy: Insight into Patient Monitoring, Erythema Nodosum Leprosum, and Bacillary Persistence  

Microsoft Academic Search

Serum IgM and IgG antibodies against Mycobacterium leprae-derived phenolic glycolipid I (PG) were determined in leprosy patients, contacts, and controls by enzyme-linked immunosorbent assay (ELISA). Anti-PG IgM levels increased from the tuberculoid (TT) to the lepromatous (LL) pole of the disease spectrum. There was a positive linear correlation between anti-PG IgM and bacillary index (BI). patients with erythema nodosum leprosum

William R. Levis; Harry C. Meeker; Georgia Schuller-Levis; Eugene Sersen; Beatrix Schwerer

1986-01-01

306

Mass spectrometric immunoassay  

SciTech Connect

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

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

2007-12-04

307

Mass spectrometric immunoassay  

DOEpatents

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

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

2013-07-16

308

Luminometry: a novel bioluminescent immunoassay enhances the quantitation of mucosal and systemic antibody responses  

Microsoft Academic Search

We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which

Raymond J. Jackson; Kohtaro Fujihashi; Hiroshi Kiyono; Jerry R. McGhee

1996-01-01

309

Simultaneous Detection of Antibodies to Six Nonhuman-Primate Viruses by Multiplex Microbead Immunoassay  

Microsoft Academic Search

To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and

Imran H. Khan; Sara Mendoza; JoAnn Yee; Matthew Deane; Kodumudi Venkateswaran; Shan S. Zhou; Peter A. Barry; Nicholas W. Lerche; Paul A. Luciw

2006-01-01

310

Is microparticle enzyme-linked immunoassay (MEIA) reliable for use in tacrolimus TDM? Comparison of MEIA to liquid chromatography with mass spectrometric detection using longitudinal trough samples from transplant recipients.  

PubMed

In the larger transplant centers where technical expertise is available, liquid chromatography with mass spectrometric detection (LC-MS) for tacrolimus therapeutic drug monitoring is replacing the popular microparticle enzyme-linked immunoassay (MEIA) as a cost-effective alternative technology. As more labs convert to LC-MS, the accuracy, precision, selectivity, and sensitivity of the tacrolimus MEIA are being challenged, using data from large populations of clinical samples. However, little attention has been paid to how the results of particular procedures may differ within and among individual patients and to how such differences may relate to patients' characteristics or to relevant biochemical parameters. So, after validation of an LC/MS procedure and verification of an LC/MS/MS procedure, the author analyzed 552 serial trough blood tacrolimus samples, collected from 38 patients over a 3-month period, by controlled MEIA and LC-MS procedures. Corresponding hematocrit and serum albumin level data were obtained. In an attempt to investigate whether the observations of others who studied population-based data could be illustrated for individuals, longitudinal data from several patients were plotted to visually elucidate any relations between the tacrolimus concentration and biochemical data. Finally tacrolimus concentration, hematocrit, and serum albumin data were compared using data stratified by transplant type, in-/outpatient status, male/female gender, or period elapsed since transplant surgery. The validated/verified LC-MS procedures were shown to be much better controlled than the MEIA during the 3-month parallel comparison study period. The longitudinal data of several individuals who experienced wide changes in the biochemical parameters clearly illustrated the relation between the difference in tacrolimus concentrations determined by MEIA and LC-MS and hematocrit (and sometimes albumin). Differences between the MEIA- and LC-MS-determined tacrolimus concentration data were strongly correlated to transplant type, in-/outpatient status, gender, time elapsed since liver transplant surgery, hematocrit, and weakly to serum albumin levels. In summary, the LC-MS methods provide highly reliable and reproducible estimates of tacrolimus concentrations, whereas the performance of MEIA technology did not provide reliable long-term performance for longitudinal therapeutic drug monitoring of tacrolimus because it was effected by several inherent demographic factors and by factors that can change over time in transplant recipients. PMID:16885716

Napoli, Kimberly L

2006-08-01

311

[Enzyme immunoassay of (24R)-brassinosteroids].  

PubMed

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide. PMID:17682395

Khripach, V A; Sviridov, O V; Priadko, A G; Litvinovskaia, R P; Drach, S V; Matveentsev, V D; Novik, T V; Mikha?lopulo, K I; Zhabinski?, V N; Zavadskaia, M I; Aver'kova, M A; Drachenova, O A; Chashchina, N M

312

Automated chemiluminescence immunoassay measurements  

NASA Astrophysics Data System (ADS)

Chemiluminescence (CL) detection offers potential for high sensitivity immunoassays (CLIAs). Several approaches were attempted to automate CL measurements. Those include the use of photographic film, clear microtitration plates, and magnetic separation. We describe a photon counting detection apparatus that performs (CLIA) measurements. The CL detector moves toward a disposable reaction vessel to create a light-tight seal and then triggers and integrates a CL signal. The capture uses antibody coated polystyrene microparticles. A porous matrix, which is a part of a disposable reaction tray, entraps the microparticle-captured reaction product. The CL signal emanated off the immune complex immobilized by the porous matrix is detected. The detection system is a part of a fully automated immunoassay analyzer. Methods of achieving high sensitivities are discussed.

Khalil, Omar S.; Mattingly, G. P.; Genger, K.; Mackowiak, J.; Butler, J.; Pepe, C.; Zurek, T. F.; Abunimeh, N.

1993-06-01

313

[Serum antibody profiles against gangliosides and sulfatide in peripheral neuropathies: evaluation of a new immunoassay].  

PubMed

We evaluated the analytical performances of a new line immunoassay (LIA) for the simultaneous detection of twelve anti-ganglioside and anti-sulfatide autoantibodies from Generic Assays, in comparison with our dot immunoassay. The LIA detected IgG and/or IgM autoantibodies against human GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b and sulfatide. Forty sera of patients with IgG autoantibody profiles in acute autoimmune neuropathies and 39 sera of patients with IgM autoantibody profiles in chronic autoimmune neuropathies were tested. To have a better sensitivity, 20 ?L of sera instead of 10 ?L were used. In these conditions, we observed a good concordance of IgG and IgM autoantibody profiles by both immunoassays. The test including novel gangliosides was easy to perform and superior for identifying autoimmune neuropathies. The data indicate that the test provides reliable simultaneous detection of autoantibodies against a large panel of human gangliosides and sulfatide with a good diagnostic usefulness in combination with clinical and electrophysiological data. PMID:21159581

Caudie, Christiane; Vial, Christophe; Petiot, Philippe; Bouhour, Françoise

314

Mass Spectrometric Immunoassay Revisited  

NASA Astrophysics Data System (ADS)

The progressive understanding and improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), realized over the years through the considerable efforts of Dr. Marvin Vestal, have made possible numerous comparable efforts involving its application in the biological sciences. Here we revisit the concepts behind one such analytical approach, Mass Spectrometric Immunoassay, which is designed to selectively detect and quantify proteins present in biological milieu.

Nelson, Randall W.; Borges, Chad R.

2011-06-01

315

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

316

Homogeneous Immunoassays: Historical Perspective and Future Promise  

NASA Astrophysics Data System (ADS)

The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

Ullman, Edwin F.

1999-06-01

317

Protein Microchips: Use for Immunoassay and Enzymatic Reactions  

Microsoft Academic Search

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 × 100 × 20-?m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen–antibody interactions within the gel. Protein microchips were used in immunoassays for detection

Pavel Arenkov; Alexander Kukhtin; Anne Gemmell; Sergey Voloshchuk; Valentina Chupeeva; Andrei Mirzabekov

2000-01-01

318

A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies  

Microsoft Academic Search

Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzy- matic tracer was obtained by coupling AMB via its amino group to

SOPHIE MACHARD; FREDERIC THEODORO; HENRI BENECH; JEAN-MARC GROGNET; ERIC EZAN

2000-01-01

319

Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips  

SciTech Connect

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

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

2008-10-20

320

Substrate-labeled fluorescent immunoassay for phenobarbital.  

PubMed

An assay for the anticonvulsant drug phenobarbital (PB) has been developed that is based on the principles of the substrate-labeled fluorescent immunoassay. A fluorogenic enzyme substrate, galactosyl umbelliferone, was covalently linked to a derivative of PB. The labeled drug, galactosyl umbelliferone-PB (GUPB), is nonfluorescent under conditions of the assay; however, hydrolysis of the galactosyl moiety by bacterial beta-galactosidase yields a fluorescent product. When GUPB is bound by antibody to PB, it is not a substrate for enzymatic hydrolysis. Thus, only GUPB not bound to antibody is hydrolyzed. In competitive binding reactions, using a fixed concentration of GUPB and a limiting amount of antibody, the PB in serum and the GUPB compete for antibody-binding sites. The fluorescence produced upon enzymatic hydrolysis of unbound GUPB is directly proportional to the concentration of PB. Unknown serum levels of PB are determined from a standard curve of fluorescent intensity versus standard PB concentrations. The assay is specific, sensitive, and easy to perform. It is carried out by adding the equivalent of 2 microliters of serum standard or unknown directly to a cuvette containing 3 ml of a buffered solution of antibody and enzyme. One-hundred microliters of GUPB is added, and the fluorescence intensity is measured after a fixed time (any time from 5 to 90 min). Using clinical specimens, our assay correlated well with a commercial enzyme immunoassay (correlation coefficient = 0.97) and had an interassay precision of less than 7%. PMID:7013165

Krausz, L M; Hitz, J B; Buckler, R T; Burd, J F

1980-01-01

321

Electrochemiluminescence flow injection immunoassay for atrazine.  

PubMed

Antibodies to atrazine were labelled with glucose oxidase and used in colorimetric enzyme linked immunosorbent assays. Transparent aminosilanized indium tin oxide coated glass electrodes were derivatized with aminodextran covalently modified with atrazine caproic acid. The labelled antibodies were used to investigate the derivatized electrodes colorimetrically and the electrodes were use in an electrochemiluminescence flow injection analyser. Electrochemiluminescence immunoassay for atrazine in the range 0-10 ppb showed that it was possible to detect less than 0.1 ppb, the precautionary limit for pesticides in drinking water recommended by the European Commission. PMID:9178513

Wilson, R; Barker, M H; Schiffrin, D J; Abuknesha, R

1997-01-01

322

Mass spectrometric immunoassay  

SciTech Connect

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin from the venoms of the prairie rattlesnake, Crotalus virdis virdis, and the Mojave rattlesnake, Crotalus scutulatus scutulatus. 18 refs., 8 figs.

Nelson, R.W.; Krone, J.R.; Bieber, A.L.; Williams, P. [Arizona State Univ., Tempe, AZ (United States)

1995-04-01

323

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

324

Morphological resonances for multicomponent immunoassays  

NASA Astrophysics Data System (ADS)

An immunoassay technique capable of detecting and identifying a number of species of microorganisms in a single analysis is described. The method uses optical-resonance size discrimination of microspheres to identify antibodies to which stained microorganisms are bound.

Whitten, W. B.; Shapiro, M. J.; Ramsey, J. M.; Bronk, B. V.

1995-06-01

325

Nanomaterials for Immunosensors and Immunoassays  

Microsoft Academic Search

\\u000a There is a continuously increasing demand for the specific and sensitive ­determination of trace amounts of analytes in complex\\u000a matrices for various ­purposes. In this respect, immunoassays and immunosensors that rely on ­antibody–antigen binding provide\\u000a a promising approach of analysis for their remarkable specificity and sensitivity. High specificity of immunoassays and immunosensors\\u000a is achieved solely by the molecular recognition of

Huangxian Ju; Xueji Zhang; Joseph Wang

326

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

327

The IGM Project: Searching For IGM Emission Over 0  

NASA Astrophysics Data System (ADS)

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

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

2013-01-01

328

Combined determination of Coxiella burnetii-specific immunoglobulin M (IgM) and IgA improves specificity in the diagnosis of acute Q fever.  

PubMed Central

Immunoglobulin M (IgM) and IgA responses in patients with acute Q fever were compared by enzyme-linked immunosorbent assay. An increase in both IgM and IgA was observed in paired sera from all 19 patients with acute Q fever, and both IgM and IgA levels showed good correlation with complement fixation test titers. Paired sera from 23 patients with infections other than Q fever were also tested. IgM levels were elevated in three of these patients, while IgA levels were elevated in three different patients (87% specificity for either IgM or IgA). As no patients in the disease control group showed elevated levels of both IgM and IgA, definition of a positive result as elevated levels of both IgM and IgA improved specificity to 100% without a decrease in sensitivity. This study indicates that detection of specific IgA is a useful adjunct to that of IgM in the diagnosis of acute Q fever.

Devine, P; Doyle, C; Lambkin, G

1997-01-01

329

Coxsackie-B-virus-specific IgM responses in patients with cardiac and other diseases.  

PubMed

An enzyme-linked immunosorbent assay (ELISA) test using polyvalent antigens and antisera was developed to detect Coxsackie-B-virus-specific IgM responses. The sera of 24 of 64 (37.5%) patients with acute pericarditis and 14 of 38 (36%) with acute myocarditis were positive for Coxsackie-B-virus-specific IgM. 4 of 30 (13.3%) patients with acute ischaemic heart disease and 2 of 28 (7.1%) patients with congestive cardiomyopathy were also positive. Coxsackie-B-virus-specific IgM was detected in the sera of 21 of 57 (36.8%) patients with Bornholm disease and 2 of 4 patients with acute-onset juvenile diabetes. Coxsackie-B-virus-specific IgM responses persisted for 6-8 weeks. Sera from patients with chronic valvular heart disease, Mycoplasma pneumoniae infections, and virus infections caused by viruses other than Coxsackie-B viruses were all negative. False-positive results did not occur when sera containing high titres of rheumatoid factor were tested. PMID:6107769

El-Hagrassy, M M; Banatvala, J E; Coltart, D J

1980-11-29

330

Long Term Storage of Antibody Sensitized Millititer (Trademark) Immunoassay Plates for the Identification and Quantitation of Francisella tularensis.  

National Technical Information Service (NTIS)

In this report we have described a modified procedure for 'sandwich' fluorogenic enzyme-linked immunosorbent assay (FELISA) for the detection and quantitation of antigen, in which immunoassay plates were pre-coated with capture antibody or, alternatively,...

R. E. Fulton Y. M. Siddiqui

1989-01-01

331

Long Term Storage of Antibody Sensitized Millititer (Trademark) Immunoassay Plates for the Identification and Quantitation of Francisella Tularensis.  

National Technical Information Service (NTIS)

In this report we have described a modified procedure for the 'sandwich' fluorogenic enzyme-linked immunosorbent assay (FELISA) for the detection and quantitation of antigen, in which immunoassay plates were pre-coated with capture antibody or, alternativ...

Y. M. Siddiqui R. E. Fulton

1989-01-01

332

Method and Apparatus for Gel Electrophoretic Immunoassay.  

National Technical Information Service (NTIS)

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic s...

A. E. Herr A. K. Singh D. J. Throckmorton

2005-01-01

333

Detection of ricin toxin in water using immunoassays  

Microsoft Academic Search

Two types of immunoassays were employed to determine ricin toxin through the substitute of A chain sub-unit in water. Protocol for detecting ricin by commercial enzyme-linked immunosorbent assay (ELISA) kits was established and evaluated. Final results were obtained by this assay in six hours and the detection limit was found to be less than 3 ng mL based on ricin

Junqi Yue; Lifeng Zhang; Zhaoguang Yang

2009-01-01

334

Optimal Design for ELBA and Other Forms of Immunoassay  

Microsoft Academic Search

In enzyme-linked immunosorbent assay (ELISA), as well as in many other kinds of immunoassay, a log-logistic or similar-shaped calibration curve is fit using standards at a series of known levels and then used to transform the measured values for the unknowns into estimated concentrations. The choice of the number of standards, the concentration of the standards, and the number of

David M. Rocke; Geoffrey Jones

1997-01-01

335

A monoclonal antibody-linked immunoassay for hemoglobin H disease  

Microsoft Academic Search

Summary A murine monoclonal antibody (mAb) was generated that recognizes hemoglobin (Hb) H, the tetrameric form (ß4) of human ß-globin chains. The antibody ß4-1 (?1, ?) does not react with Hbs A, F, Bart's, or isolated ß chains, indicating that the antibody recognizes an epitope comprised of multiple ß chains. A simple, rapid, and sensitive enzyme immunoassay was established to

M. Shyamala; C. R. Kiefer; H. Moscoso; F. A. Garver

1992-01-01

336

Development and Persistence of West Nile Virus-Specific Immunoglobulin M (IgM), IgA, and IgG in Viremic Blood Donors  

PubMed Central

West Nile Virus (WNV) antibody development and persistence were investigated in blood donors who made WNV RNA-positive (viremic) donations in 2003. Plasma samples from the index donations and follow-up serum or plasma samples were tested for WNV immunoglobulin M (IgM), IgA, and IgG by using enzyme-linked immunosorbent assays. Antibody development was investigated with 154 samples collected from 84 donors 1 to 21 days after their RNA-positive, antibody-negative, index donation. WNV IgM and IgA were first detected on day 3, and all samples collected after day 9 were WNV IgM and IgA positive; WNV IgG was first detected on day 4, and all samples collected after day 16 were positive. Antibody persistence in this donor group (index donations antibody negative) was evaluated by using 128 samples collected from 89 donors on days 22 to 440 of follow-up; 88% of samples were WNV IgM positive, 86% were WNV IgA positive, and 100% were WNV IgG positive. In linear regression analysis, trendlines for WNV IgM and IgA reached the value discriminating positive from negative results at 218 days and 232 days of follow-up, respectively. Similar WNV IgM and IgA persistence trends characterized 27 donors whose index samples were positive for WNV IgM and IgA, as well as 14 donors whose index samples were positive for WNV IgG but negative for WNV IgM. These findings show that WNV IgG emerges after WNV IgM and IgA and that both WNV IgM and IgA typically persist for at least 6 months after infection. Thus, unlike some other flavivirus infections, WNV infection is not characterized by a relatively rapid disappearance of virus-specific IgA.

Prince, Harry E.; Tobler, Leslie H.; Lape-Nixon, Mary; Foster, Gregory A.; Stramer, Susan L.; Busch, Michael P.

2005-01-01

337

Ender Bir IgM Nefropati Nedeni: Eriflkin Still Hastal›¤› Rare Cause of IgM Nephropathy: Adult Still's Disease  

Microsoft Academic Search

The type of mesengial proliferative glomerulonephritis having IgM deposition in DIF examination is called IgM nephropathy. A case performed renal biopsy due to urine findings and diagnosed as IgM nephropathy is presented. The patient was hospitalized for a 3-week fever of 39°C and weight loss. Viral and bacterial causes were not positive, and ANA, anti-ANA and ANCA were negative. Owing

Suat Ünver; Aptullah Haholu; Yaflar Küçükardal; Enes Murat Atasoyu

338

Performance of hepatitis B assays on the Bayer ADVIA Centaur Immunoassay System.  

PubMed

Bayer HealthCare LLC, Diagnostics Division, has developed several new assays on the ADVIA Centaur immunoassay system for the detection of markers of hepatitis B virus infection in human serum and plasma. This panel includes assays for: hepatitis B surface antigen (HBsAg), a confirmatory test method for HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). These assays employ magnetic particle separation technology with direct chemiluminescence for optimal assay performance. All of the assays are fully automated, require sample volumes ranging from 15 microl to 100 microl (with the exception of the ADVIA Centaur HBsAg Confirmatory Assay, which requires 2 x 100 microl), and have throughputs of up to 240 tests per hour. The five ADVIA Centaur HBV assays were tested in extensive performance evaluations conducted at two sites in Europe. The performance evaluations, which included samples from HBV-infected individuals, blood donors, hospitalized/clinical patients, and HBV vaccinees (for Anti-HBs evaluation), generated performance data in support of obtaining the Communautés Européennes (CE) mark for European market distribution. The HBV performance evaluations resulted in an overall diagnostic specificity > 99%, i.e. 99.94% for the ADVIA Centaur HBsAg Assay, 100% for the ADVIA Centaur Anti-HBs Assay, 100% for the ADVIA Centaur HBc IgM Assay and 99.94% for the ADVIA Centaur HBc Total Assay. All of the ADVIA Centaur assays showed a very good diagnostic sensitivity on these populations with 100% for the ADVIA Centaur HBsAg Assay, 99.0% for the ADVIA Centaur Anti-HBs Assay, 98.53% for the ADVIA Centaur HBc IgM Assay and 100% for the ADVIA Centaur HBc Total Assay. The ADVIA Centaur HBsAg Confirmatory Test confirmed 100% of the positive HBsAg samples. Testing of interfering substances and potential cross-reacting samples for all ADVIA Centaur HBV assays resulted in no change in interpretation of the results. Assay performance was further evaluated using HBV seroconversion panels with comparable or better results when compared to the comparison assays. The performance evaluation data demonstrate that the ADVIA Centaur HBV assays are specific and sensitive automated immunoassays for detection of antigens and antibodies to hepatitis B virus with performance that is comparable to those of currently marketed assays. Additionally, these assays have the advantage of being available on the ADVIA Centaur immunoassay system, which provides for the flexibility of high throughput and full automation. PMID:15000222

van Helden, Josef; Denoyel, Gérard; Karwowska, Sylwia; Reamer, Randy; Schmalz, John; Wright, Ted; Preisel-Simmons, Barbara

2004-01-01

339

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...

340

Development of an enzyme-immunoassay (EIA) for the measurement of follicle-stimulating-hormone (FSH) in Callitrichid primates using a monoclonal antibody against the human-FSH-beta-subunit.  

PubMed

Despite the importance of Callitrichid primates in both biomedical and conservation research, practical and reliable immunoassays for the measurement of follicle-stimulating hormone (FSH) have not yet been described. A panel of monoclonal antibodies against specific peptide fragments within either the alpha or beta subunit of human FSH was evaluated for their ability to recognize FSH from Callitrichid and other New World primates. One of these, monoclonal antibody 46.3h6.b7 raised against human FSH, was selected due to its ability to recognize marmoset monkey FSH and its low crossreactivity with other gonadotrophins. The antibody formed the basis of an enzymeimmunoassay using a highly purified human urinary FSH (Metrodin, Serono) preparation coupled to biotin as label and unmodified as standard. After 24 h incubation, antibody bound label was visualized by addition of streptavidin-peroxidase followed by the appropriate substrate. Parallelism was obtained between the standard and dilutions of pituitary extracts, urine and plasma from the common marmoset (Callithrix jacchus) as well as from two tamarin species (Saguinus fuscicollis and S. oedipus) and one squirrel monkey (Saimiri sciureus). Profiles of plasma and urinary FSH during the follicular phase are shown for two individual marmosets. The ability to measure FSH in Callitrichidae provides new opportunities for studies of the reproductive biology of these New World primate species. PMID:9057964

Rosenbusch, J; Dias, J A; Hodges, J K

1997-01-01

341

IgM in Microbial Infections: Taken for Granted?  

PubMed Central

Much has been learned about the structure, function, and production of IgM, since the antibody's initial characterization. It is widely accepted that IgM provides a first line of defense during microbial infections, prior to the generation of adaptive, high affinity IgG responses that are important for long-lived immunity and immunological memory. Although IgM responses are commonly used as a measure of exposure to infectious diseases, it is perhaps surprising that the role of and requirement for IgM in many microbial infections has not been well explored in vivo. This is in part due to the lack of capabilities, until relatively recently, to evaluate the requirement for IgM in the absence of coincident IgG responses. Such evaluations are now possible, using gene-targeted mouse strains that produce only IgM, or isotype-switched IgG. A number of studies have revealed that IgM, produced either innately, or in response to antigen challenge, plays an important and perhaps underappreciated role in many microbial infections. Moreover, the characterization of the roles of various B cell subsets, in the production of IgM, and in host defense, has revealed important and divergent roles for B-1a and B-1b cells. This review will highlight studies in which IgM, in its own right, has been found to play an important role, not only in early immunity, but also in long-term protection, against a variety of microbial pathogens. Observations that long-lived IgM responses can be generated in vivo suggest that it may be feasible to target IgM production as part of vaccination strategies.

Racine, Rachael; Winslow, Gary M.

2009-01-01

342

Increased levels of serum IgM antibody to staphylococcal enterotoxin B in patients with rheumatoid arthritis.  

PubMed Central

OBJECTIVE--To investigate the role of superantigen in rheumatoid arthritis (RA) by assaying the serum levels of staphylococcal enterotoxin B (SEB) antibodies. METHODS--Serum IgG and IgM SEB antibodies were measured using an enzyme linked immunosorbent assay (ELISA), and confirmed by Western blot analysis. The T cell receptor V beta (TCR V beta) repertoire was analysed using the reverse transcriptase polymerase chain reaction. RESULTS--RA patients had increased levels of serum IgM SEB antibody compared with normal subjects, patients with systemic lupus erythematosus, Sjögren's syndrome, and Behçet's disease. The titres of rheumatoid factor (RF) showed no correlation with the levels of IgM SEB antibodies, and the levels of SEB antibodies were not inhibited by the addition of human immunoglobulin, or after absorption of RF. RA patients whose disease duration was less than 10 years had greater levels of serum IgM SEB antibodies than those with disease duration more than 10 years. The levels of IgM and IgG SEB antibodies in synovial fluid from RA patients were correlated with those in their sera. Western blot analysis detected IgM and IgG SEB antibodies as a band of approximately 30 kDa molecular size. The percentage of TCR V beta 2, V beta 5.2, and V beta 12 in phytohaemagglutinin stimulated peripheral T cells correlated significantly with the levels of serum IgM SEB antibody in RA patients. CONCLUSION--These results suggest that SEB, one of the superantigens, may have a critical role in the pathogenesis of RA. Images

Origuchi, T; Eguchi, K; Kawabe, Y; Yamashita, I; Mizokami, A; Ida, H; Nagataki, S

1995-01-01

343

Solid supports for microarray immunoassays  

Microsoft Academic Search

Stimulated by the achievements of the first phase in genomics and the resulting need of assigning functions to the acquired sequence information, novel formats of immunoassays are being developed for high- throughput multi-analyte studies. In principle, they are similar in nature to the microarray assays already established at the level of nucleic acids. However, the biochemical diversity and the sheer

Wlad Kusnezow

2003-01-01

344

Performance of Opus immunoassays for thyroxine and beta-human chorionic gonadotrophin in serum.  

PubMed

Two representative immunoassays for measuring thyroxine and beta-subunit of human chorionic gonadotrophin in serum, using the Opus immunoassay analyzer, were evaluated by comparing them to the reference RIA for T4 and beta-HCG enzyme immunoassay. Both assays were superior in accuracy and precision than the reference methods and exhibited good linearity throughout the concentration range needed for discriminating abnormally low and elevated concentrations from the established reference ranges of thyroxine and beta-human chorionic gonadotrophin in serum. Correlation between the results of the Opus immunoassays and the reference assays for T4 and beta-HCG was very good with correlation coefficients of 0.92 and 0.98, respectively. PMID:8951607

Murthy, V V

1996-01-01

345

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

346

Duplex Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies  

Microsoft Academic Search

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne

Alison J. Johnson; Ronald C. Cheshier; Giorgio Cosentino; Robert S. Lanciotti; Alicia A. Johnson; Brad J. Biggerstaff; Denise A. Martin; Amanda J. Panella; Olga Kosoy

2005-01-01

347

Comparison of Three Automated Immunoassay Methods for the Determination of Epstein-Barr Virus-Specific Immunoglobulin M ?  

PubMed Central

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

Berth, Mario; Bosmans, Eugene

2010-01-01

348

Multiplex immunoassay platforms based on shape-coded poly(ethylene glycol) hydrogel microparticles incorporating acrylic acid.  

PubMed

A suspension protein microarray was developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles for potential applications in multiplex and high-throughput immunoassays. A simple photopatterning process produced various shapes of hydrogel micropatterns that were weakly bound to poly(dimethylsiloxane) (PDMS)-coated substrates. These micropatterns were easily detached from substrates during the washing process and were collected as non-spherical microparticles. Acrylic acids were incorporated into hydrogels, which could covalently immobilize proteins onto their surfaces due to the presence of carboxyl groups. The amount of immobilized protein increased with the amount of acrylic acid due to more available carboxyl groups. Saturation was reached at 25% v/v of acrylic acid. Immunoassays with IgG and IgM immobilized onto hydrogel microparticles were successfully performed with a linear concentration range from 0 to 500 ng/mL of anti-IgG and anti-IgM, respectively. Finally, a mixture of two different shapes of hydrogel microparticles immobilizing IgG (circle) and IgM (square) was prepared and it was demonstrated that simultaneous detection of two different target proteins was possible without cross-talk using same fluorescence indicator because each immunoassay was easily identified by the shapes of hydrogel microparticles. PMID:22969408

Park, Saemi; Lee, Hyun Jong; Koh, Won-Gun

2012-06-20

349

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

2013-03-11

350

Recent enterovirus infection in type 1 diabetes: evidence with a novel IgM method.  

PubMed

Enterovirus (EV) infection has been associated with Type 1 (T1D) diabetes and on a few occasions virus could be isolated at onset of the disease. Using two such isolates as antigens in a quantitative PCR enhanced immunoassay (T1D-EV-QPIA) we have measured IgM antibodies against such potentially diabetogenic viruses in serum from 33 newly diagnosed T1D children, 24 siblings, and 27 healthy children. Sera were also analysed with regard to autoantibodies against GAD65, the cytokine TNF-alpha and the chemokine IP-10. EV-RNA detection was performed on peripheral blood mononuclear cells (PBMC). IgM antibodies against this "new" EV antigen were more frequent in serum from T1D children than in serum from siblings and/or controls (P < 0.001). EV-RNA was detected more frequently in PBMC from T1D children than in healthy control children (P < 0.001) and also compared to the siblings (P < 0.003). The cytokine TNF-alpha was less frequently detected in serum from the T1D children compared with serum from siblings and/controls (P < 0.001). A positive correlation was found between the results obtained with the T1D-EV-QPIA and the EV-PCR (P < 0.001). These findings are in line with earlier findings of an increased frequency of enteroviral infections in newly diagnosed T1D patients. In addition, we found that T1D children at onset of the disease had lower frequencies of the chemokine TNF-alpha in their serum than age- and sex-matched controls had, suggesting an impaired immune response. PMID:17935175

Elfaitouri, A; Berg, A-K; Frisk, G; Yin, H; Tuvemo, T; Blomberg, J

2007-12-01

351

Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.  

PubMed

As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa. PMID:20162378

Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

2010-02-18

352

Intrafamilial correlation analysis for IgM serum levels.  

PubMed Central

The IgM serum level was determined in the members of 29 healthy families. The IgM mean concentrations between fathers and mothers and between sons and daughters were significantly different (P less than .01), with higher serum IgM levels in females than in males. Simple linear regression analysis was done for the following intrafamilial combinations: son-father, daughter-father, son-mother, and daughter-mother. Significant correlation coefficients (P less than .05) were obtained in all four combinations, which does not support the X-linked gene hypothesis (i.e., that the X chromosome carries quantitative genes for immunoglobulin M). An alternative explanation for the differences between sexes for IgM serum concentration is considered.

Guizar-Vazquez, J; Saint-Martin, F P; Rostenberg, I; Suarez, P E; Armendares, S

1977-01-01

353

Analytical validation of anti-toxoplasma IgG immunoassays.  

PubMed

There are often discrepancies when using different methods to measure anti-Toxoplasma gondii IgG levels in patient samples. The diagnostic performance of a chemiluminescent immunoassay (CLIA) and an enzyme-linked fluorescent assay (ELFA) used as confirmatory tests for samples identified as positive or equivocal by an electrochemiluminescent immunoassay (ECLIA) were examined. Cut-off values were those stated by the manufacturer, and Western blot was used to confirm the results of all methods. All samples identified as positive by ECLIA (n=93) were confirmed as positive by Western blot, as were 14 of the 28 samples identified as equivocal. When these 121 samples were retested, the sensitivities of CLIA and ELFA were 64.4% and 73.8%, respectively. Both methods exhibited a specificity of 100%. This study confirms that the results obtained from the different immunoassays are not comparable, and neither CLIA nor ELFA should be used to confirm ECLIA results, which should instead be confirmed by methods such as Western blot or Sabin-Feldman dye test. PMID:23141993

Souza, Guenael Freire de; Carvalho, Darlene; Pedrosa, William; Franck, Jacqueline; Piarroux, Renaud

2012-11-08

354

Prevalence of Legionella-specific IgG and IgM Antibody in a Dental Clinic Population  

Microsoft Academic Search

This study was undertaken to determine the frequency of Legionella infection in a dental clinic setting. Serum samples from 270 dental clinic personnel were evaluated using an enzyme-linked immunosorbent assay to detect Legionella-specific IgM and IgG antibodies. The pooled-species whole-cell-antigen preparation used in these assays was derived from six Legionella pneumophila strains and one strain each from Legionella bozemanii and

P. G. Fotos; H. N. Westfall; I. S. Snyder; R. W. Miller; B. M. Mutchler

1985-01-01

355

Immunoassay of Mouse Immunoglobulin G by a Light-Addressable Potentiometric Sensor.  

National Technical Information Service (NTIS)

A sensitive enzyme immunoassay for the quantitation of mouse immunoglobulin G (mIgG) was developed using a light-addressable potentiometric (LAP) sensor as the detection system. The assay was carried out on nitrocellulose membrane filters and used sandwic...

H. G. Thompson W. E. Lee

1991-01-01

356

Detection of cyanobacterial toxins (microcystins) in waters of northeastern wisconsin by a new immunoassay technique  

Microsoft Academic Search

The development of reliable, sensitive immunoassay techniques for detection of microcystins in water is becoming increasingly important. We have developed an enzyme-linked immunosorbent assay (ELISA) potentially able to detect microcystins at concentrations as low as 95 pg microcystin\\/ml water. The procedure uses antibodies extracted from the eggs of immunized chickens, eliminating the need to collect blood from laboratory rabbits. The

C. M McDermott; R Feola; J Plude

1995-01-01

357

Use of bifunctional hybrid ?-lactamases for epitope mapping and immunoassay development  

Microsoft Academic Search

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis ?-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random

Andy Chevigné; Nursel Yilmaz; Gilles Gaspard; Fabrizio Giannotta; Jean Marie François; Jean Marie Frčre; Moreno Galleni; Patrice Filée

2007-01-01

358

IMMUNOASSAYS FOR METAL IONS. (R824029)  

EPA Science Inventory

Abstract Antibodies that recognize chelated forms of metal ions have been used to construct immunoassays for Cd(II), Hg(II), Pb(II), and Ni(II). In this paper, the format of these immunoassays is described and the binding properties of three monoclonal antibodies direc...

359

Natural serum IgM maintains immunological homeostasis and prevents autoimmunity  

Microsoft Academic Search

Natural (i.e. low-affinity, preimmune) IgM has a wide range of actions in the immune system. That IgM is important in defence against infection has been recognised for many years but recently, due to the generation of mouse models specifically deficient in serum IgM, other functions of serum IgM have been revealed. The participation of natural IgM in autoimmunity has been

Jessica J. Manson; Claudia Mauri; Michael R. Ehrenstein

2005-01-01

360

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats.  

PubMed

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and alpha-zearalenol (alpha-ZOL) (69%) recognition, while cross-reactivities with alpha-zearalanol, zearalanone, beta-zearalenol and beta- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 microg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 microg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and alpha-ZOL). PMID:19575188

Burmistrova, Natalia A; Goryacheva, Irina Yu; Basova, Evgenia Yu; Franki, Ann-Sophie; Elewaut, Dirk; Van Beneden, Katrien; Deforce, Dieter; Van Peteghem, Carlos; De Saeger, Sarah

2009-07-04

361

Protective Roles of Natural IgM Antibodies  

PubMed Central

Antibodies are a vital part of the armamentarium of the adaptive immune system for the fine-tuning of the recognition and response to foreign threats. However, in health there are some types of antibodies that instead recognize self-antigens and these contribute to the enhancement of primitive innate functions. This repertoire of natural IgM antibodies is postulated to have been selected during immune evolution for their contributions to critical immunoregulatory and housekeeping properties. The clearance of dying cells is one of the most essential responsibilities of the immune system, which is required to prevent uncontrolled inflammation and autoimmunity. In the murine immune system, natural IgM antibodies that recognize apoptotic cells have been shown to enhance the phagocytic clearance of dead and dying cells and to suppress innate immune signaling pathways. In the mouse, natural IgM are often the products of B-1 cell clones that arise during immune development without an absolute requirement for exogenous antigenic stimulation. In patients with systemic lupus erythematosus, IgM autoantibodies, which bind to neo-epitopes on apoptotic cells, have been demonstrated to be present at significantly higher levels in patients with lower disease activity and with less severe organ damage. While certain specificities of IgM autoantibodies correlate with protection from lupus renal disease, others may convey protective properties from lupus-associated atherosclerotic cardiovascular disease. New and unexpected insights into the functional roles of IgM antibodies are still emerging, especially regarding the functions of natural antibodies. Herein, we review recent progress in our understanding of the potential roles of natural IgM autoantibodies in the regulation of immune homeostasis and for protection from autoimmune and inflammatory diseases.

Gronwall, Caroline; Vas, Jaya; Silverman, Gregg J.

2012-01-01

362

A method for selective conjugation of an analyte to enzymes without unwanted enzyme–enzyme cross-linking  

Microsoft Academic Search

The conjugation of a ligand to an enzyme is often a necessary step in the development of enzyme-linked immunoassays. Such conjugation is typically accomplished by reacting an amine with a carboxyl functional group in the presence of an activator such as a carbodiimide. However, one enzyme’s free carboxyl groups often react with another’s free amino groups and a large amount

Vincent C Lombardi; David A Schooley

2004-01-01

363

Development and validation of a sensitive immunoassay for the skeletal muscle isoform of creatine kinase  

Microsoft Academic Search

Creatine kinase (CK) is a marker of muscle damage and pathology present as multiple tissue-specific circulating isoforms. CK is often measured using enzyme activity assays that are unable to distinguish these isoforms. We have developed an immunoassay specific for the MM isoform of CK, found predominantly in skeletal muscle, which uses very small volumes of plasma (1–2?L). A sandwich enzyme-linked

Kim R. Lo; Suzanne M. Hurst; Kelly R. Atkinson; Tom Vandenbogaerde; C. Martyn Beaven; John R. Ingram

2010-01-01

364

Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)  

NASA Astrophysics Data System (ADS)

Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this technique are a reduction in reagents, higher sensitivity, minimal preparation of complex samples such as blood, real-time calibration, and extremely rapid analysis.

Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

2000-03-01

365

Differences in IgM synthesis to gut bacterial peptidoglycan polysaccharide after burn injury and gut ischemia.  

PubMed

Both burn injury and intestinal ischemia have been proven to induce bacterial translocation from the gut. It is still unknown, however, whether the bacteria induces immune response in these different models. To assess this, we measured in vitro IgM synthesis to peptidoglycan polysaccharide (PGPS), a ubiquitous gut bacterial antigen, after burn injury or gut ischemia-reperfusion in a mouse model. Eighty-five BALB/c mice were divided into four groups. Gut ischemia was produced by placing a vessel loop around the superior mesenteric artery at celiotomy (group Isc; n = 31). After 45 minutes, the abdomen was reopened, and the vessel loop removed. All animals had visible gut ischemia. Control mice (group Isc-C; n = 15) underwent two sham operations. Burn injury was 25% body surface area full-thickness to the dorsum (group B; n = 27). Another control group (B-C; n = 12) was also used. Animals were euthanized 24 hours after recirculation or 5 days after the burn injury. All spleens were removed, and cell suspensions prepared. Cells were cultured in 2.5 micrograms/ml lipopolysaccharide for 5 days, and anti-PGPS IgM level in the supernatant was measured by an enzyme-linked immunosorbent assay. Intestinal ischemia produced a significant rise in in vitro anti-PGPS IgM synthesis per 10(5) lymphocytes, which is the principal immunoglobulin response to infection. However, anti-PGPS IgM in mice after burn injury was significantly decreased. This decreased IgM synthesis after burn injury compared to gut ischemia may represent continued immune impairment from the burn wound, and may account for organ dysfunction related to bacterial translocation after burn injury. PMID:8736368

Tabata, T; deSerres, S; Meyer, A A

366

rROP2186–533: A Novel Peptide Antigen for Detection of IgM Antibodies Against Toxoplasma gondii  

PubMed Central

Abstract Toxoplasma gondii infections are prevalent in a wide range of mammalian hosts including humans. Infection in pregnant women may cause the transmission of parasite to the fetus that makes serious problems. IgM antibodies against Toxoplasma (Toxo-IgM) have been believed to be significant indicators for both recently acquired and congenital toxoplasmosis. So far, however, there has not been any recognized protein of T. gondii that specifically reacts to IgM antibodies. Here, an antigen exclusively for detection of IgM antibodies screened by two-dimensional electrophoresis and mass spectrometry has been reported. The study identified 13 Toxoplasma proteins probed by IgG antibodies and one (rhpotry protein 2 [ROP2]) by IgM antibodies with human sera of Toxo-IgM–-IgG+ and -IgM+-IgG–, respectively, which had been prescreened by Toxo-IgM and -IgG commercial kits from the suspected cases. Following cloning, expression, and purification of the fragment of ROP2186–533, an enzyme-linked immunosorbent assay with rROP2186–533 to measure IgM and IgG antibodies was developed. As a result, 100%(48/48) of sera with Toxo-IgM+-IgG–showed positive Toxo-IgM but none of them (0%) showed positive Toxo-IgG when rROP2186–533 was used as antigen. Neither Toxo-IgG nor Toxo-IgM antibodies were found when tested with 59 sera of Toxo-IgM–-IgG+. These results indicate that rROP2186–533 could be used as an antigen that specifically capture Toxo-IgM antibodies and may have a high potential in the serological diagnosis of both acute acquired and congenital toxoplasmosis.

Liu, Lili; Liu, Tingting; Yu, Li; Cai, Yihong; Zhang, Aimei; Xu, Xiucai; Luo, Qingli; Hu, Yuansheng; Song, Wenjian; Lun, Zhaorong; Lu, Fangli; Wang, Yong

2012-01-01

367

Theoretical and practical implications of a new definition of the minimal detectable concentration for immunoassays  

NASA Astrophysics Data System (ADS)

The minimal detectable concentration (MDC), the smallest analyte concentration an immunoassay can reliably measure, is a fundamental property of an assay. Different interpretations of 'the smallest concentration an immunoassay can reliably measure' have led to different mathematical formulations of the MDC definition. We interpret this concept to mean the smallest analyte concentration the immunoassay may report as greater than the zero dose with high probability, say 0.95. Using Bayes' theorem we have developed a new MDC definition and shown that each of the current definitions approximates some component of the new one. We extend our paradigm for computing the MDC and derive a statistical framework for analyzing uncertainty in small analyte concentrations. We compute for any immunoassay measurement the probability that it exceeds the zero dose, its 95% confidence interval, its coefficient-of-variation (CV) and the probability that it is greater than any previous measurement. We illustrate the framework in a study of theoretical data based on our previous analyses of the Abbott microparticle capture enzyme immunoassay assay (MEIA) for prostate- specific antigen (PSA). The paradigm provides a sounder conceptual and computational approach for measuring reliably low concentrations of biological substances and for defining a positive result for screening and diagnostic tests.

Brown, Emery N.

1996-04-01

368

Improved serological diagnosis of Toxoplasma gondii infection by detection of immunoglobulin A (IgA) and IgM antibodies against P30 by using the immunoblot technique.  

PubMed Central

Immunoglobulin M (IgM) and IgA antibodies against the major surface protein of Toxoplasma gondii were determined in a total of 195 human sera and five human cerebrospinal fluids by using a P30 membrane extract and the immunoblot technique. By using two different T. gondii strains (RH and BK) simultaneously as antigens, we were able to demonstrate diagnostically important strain-specific human antibody responses in 4.5% of the samples tested. A comparison of the immunoblot technique with an IgM immunocapture enzyme-linked immunosorbent assay demonstrated that the IgM and IgA immunoblot seems to be of advantage in the diagnosis of acute toxoplasmosis in certain groups of patients, especially in the diagnosis of cerebral toxoplasmosis in patients with AIDS. The immunoblot technique described is easy to perform and might be useful as an additional serological assay for routine diagnosis of T. gondii infections. Images

Gross, U; Roos, T; Appoldt, D; Heesemann, J

1992-01-01

369

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.

Maragos, Chris

2009-01-01

370

Chemiluminescence Immunoassay (CLIA) for Candida Antigen.  

National Technical Information Service (NTIS)

The project demonstrated that the combination of chemiluminescent (CL) acridinium esters and monoclonal antibodies to Candida cell-wall mannan is capable of generating a simple non-isotopic immunoassay for Candida mannan in serum. The sensitivity of the i...

A. Patel

1987-01-01

371

Multiple recurrent hordeola associated with selective IgM deficiency.  

PubMed

An external hordeolum is an acute, suppurative inflammation of the glands of Zeis and sweat glands or hair follicles most commonly caused by staphylococci, usually in the setting of a chronic blepharitis.(1) We report a case of a boy with unilateral multiple recurrent hordeola in association with selective IgM deficiency. PMID:11182678

Kiratli, H K; Akar, Y

2001-02-01

372

Analysis of Oxidative Stress Biomarkers Using a Simultaneous Competitive/Non-Competitive Micromosaic Immunoassay  

PubMed Central

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 ?L sample and 45 min for completion. Logistic curve fits and LOD statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Murphy, Brian M.; Dandy, David S.

2009-01-01

373

Monoclonal Antibody Based Enzyme Immunoassay for Marihuana (Cannabinoid) Compounds  

Microsoft Academic Search

MAb against ?-THCA was produced by fusing hybridoma with splenocytes immunized with ?-THCA-BSA conjugate and hypoxanthine, aminopterine, thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. The cross-reaction of anti-?-THCA antibody against other cannabinoids was very wide, thus many cannabinoids and a spiro-compound were reactive suggesting that 2?-hydroxyl, 6?-hydroxyl or 6?-O-alkyl, 4?-alkylbenzene ring moiety is necessary for its reactivity. It became evident that

Hiroyuki Tanaka; Yuri Goto; Yukihiro Shoyama

1996-01-01

374

Evaluation of immunoassays for the measurement of insulin and C-peptide as indirect biomarkers of insulin misuse in sport: Values in selected population of athletes  

Microsoft Academic Search

Insulin and C-peptide have been proposed as possible biomarkers of human insulin hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was performed.Enzyme Amplified Sensitivity Immunoassay (EASIA) assays (Human Insulin-EASIA and C-peptide EASIA kits from BioSource) were evaluated for insulin and C-peptide in serum.The intra- and inter-laboratory precision and accuracy values were good for the

Rosario Abellan; Rosa Ventura; Ilaria Palmi; Simonetta di Carlo; Rita di Giovannandrea; Montse Bellver; Ramon Olive; Jose Antonio Pascual; Roberta Pacifici; Jordi Segura; Piergiorgio Zuccaro; Simona Pichini

2009-01-01

375

Comparison of immunoassay and gas chromatography\\/mass spectrometry methods for measuring 3,5,6-trichloro-2-pyridinol in multiple sample media  

Microsoft Academic Search

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. The food and urine samples were analyzed by a laboratory-based 96-microwell plate immunoassay format. Methanol was used as the extraction solvent for the

Jane C. Chuang; Jeanette M. Van Emon; Allen W. Reed; Nadia Junod

2004-01-01

376

Layer-by-layer hydroxymethyl ferrocene modified sensor for one-step flow\\/stop-flow injection amperometric immunoassay of ?-fetoprotein  

Microsoft Academic Search

A rapid one-step flow\\/stop-flow injection amperometric immunoassay for ?-fetoprotein (AFP) using a novel home-produced electrochemical sensor was proposed. The sensor was prepared using layer-by-layer adsorption of positively charged poly(allylamine) (PAA) and negatively charged hydroxymethyl ferrocene on a screen-printed electrode (SPE). The electrochemistry of the immobilized ferrocene moieties showed a surface-controlled electrode process. Based on an electrochemical enzyme-linked immunoassay with the

Zong Dai; Simona Serban; Huangxian Ju; Nabil El Murr

2007-01-01

377

Multiplex detection of plant pathogens using a microsphere immunoassay technology.  

PubMed

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

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

2013-04-26

378

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

2013-01-04

379

IgM, Fc?-receptors and malarial immune evasion  

PubMed Central

"This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org." Immunoglobulin M (IgM) is an ancestral antibody class found in all jawed vertebrates from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied, and most enigmatic of the vertebrate immunoglobulins has recently been shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. Here we discuss how this association might come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just discovered Fc?-receptor (FcµR) localized to B and T cell surfaces. As this parasite may exploit an interaction with IgM to not only limit immune detection but also manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies.

Czajkowsky, Daniel M.; Salanti, Ali; Ditlev, Sisse B; Shao, Zhifeng; Ghumra, Ashfaq; Rowe, J. Alexandra; Pleass, Richard J

2010-01-01

380

Evaluation of a Multiplex Flow Immunoassay for Detection of Epstein-Barr Virus-Specific Antibodies  

Microsoft Academic Search

Conventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immu- nofluorescence assay (IFA) and enzyme immunoassay (EIA). While sensitive and specific, these methods are labor-intensive and require separate assays for each analyte. This study evaluated the performance of a multiplex bead assay (BioPlex 2200; Bio-Rad Laboratories, Hercules, CA) for the simultaneous detection of immunoglobulin G (IgG) and

M. J. Binnicker; D. J. Jespersen; J. A. Harring; L. O. Rollins; E. M. Beito

2008-01-01

381

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

382

Oral fluid for the detection of drugs of abuse using immunoassay and LC-MS/MS.  

PubMed

The utility of oral fluid as a sample matrix for the analysis of drugs has been increasing in popularity over the last few years. This is largely because of collection advantages over other matrices, but also due to the rapid improvements in analytical assays including highly sensitive liquid reagent format enzyme immunoassays and LC-MS/MS. This review will highlight improvements in assay formats, sensitivity, laboratory equipment and sample processing using low sample volumes to expand drug test profiles. PMID:23795933

Moore, Christine; Crouch, Dennis

2013-06-01

383

Detection of Molecular signatures of life using immunoassay techniques  

NASA Astrophysics Data System (ADS)

The Miniaturized Array for Solar System Exploration (MASSE) will use a microarray of antibody assays to search for biomarkers in extraterrestrial environments. We have now used enzyme linked immunosorbent assay (ELISA) to demonstrate the feasibility of immuno-detection of biomarkers in terrestrial soil, JSC-1 Mars regolith simulant, and terrestrial polar permafrost as analogues f ro extraterrestrial materials. We have also demonstrated that the technique works at microgravity and Martian gravity. Studies are now underway to test immunoassay techniques and antibody arrays at varying pressures and temperatures. It is expected that these studies will lead to a flight ready biomarker detection instrument that will be landed and operated on the Martian surface in 2009.

McKay, D.; Steele, A.; Warmflash, D.; Maule, J.; Lynch, K.

384

Development of an ultrasensitive immunoassay for detecting tartrazine.  

PubMed

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

2013-06-25

385

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.

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

2013-01-01

386

Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices.  

PubMed

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution. PMID:18783243

Manclús, Juan J; Moreno, María J; Plana, Emma; Montoya, Angel

2008-09-11

387

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.

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

388

Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection.  

PubMed

In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3?) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay. PMID:21348438

Liu, Rui; Liu, Xing; Tang, Yurong; Wu, Li; Hou, Xiandeng; Lv, Yi

2011-02-24

389

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, Angel; Manclús, Juan J

2013-08-03

390

The IgM Response of Children to Salmonella Typhosa Vaccine. I. Measurements of Anti-Typhoid Concentrations and Their Proportions of Total Serum IgM Globulins.  

National Technical Information Service (NTIS)

Eight children immunized with commercial typhoid vaccine had elevations of total IgM globulins that averaged 55% above pre-immunization levels within 10 days. Concentrations of IgM specific for the surface antigens of Salmonella typhosa were found by meas...

W. A. Altemeier J. A. Bellanti E. L. Buescher

1969-01-01

391

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...

392

SQUID based remanence measurements for immunoassays  

Microsoft Academic Search

The use of fine magnetic particles as labels for antibodies and the measurement of their remanent magnetization for the preparation of immunoassays is presented. Antibodies were coupled with magnetic nanoparticles and samples were prepared by reaction of the magnetically labeled antibodies with their solid phase adsorbed antigen. After exposing the samples to a field of some mT a dc-SQUID system

R. Kotitz; H. Matz; L. Trahms; H. Koch; W. Weitschies; T. Rheinlander; W. Semmler; T. Bunte

1997-01-01

393

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

PubMed

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

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

2012-07-25

394

Peptide ligands that bind IgM antibodies and block interaction with antigen.  

PubMed

We have selected a peptide-display phage library on IgM Abs and identified a panel of phage-expressing peptides that bind to IgM Abs in general, but not to Abs of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM Abs. The peptides bind to both soluble pentameric Abs and to monomeric cell-surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM Abs to Ag. These peptides may create confounding artifacts when IgM Abs are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility. PMID:10395676

Glee, P M; Pincus, S H; McNamer, D K; Smith, M J; Burritt, J B; Cutler, J E

1999-07-15

395

The structure of two IgMs showing different activity from a patient with Waldenstrom's macroglobulinaemia.  

PubMed Central

The two monoclonal IgMs (IgM1 and IgM2) were characterized from a patient Waldenström's macroglobulinaemia that resulted in a gammapathy. Heavy and light chains were isolated from the IgM. The complete primary structure of the two light chains and the NH2-terminal region of the two heavy chain molecules were determined. The sequence data indicated that the heavy and light chains from both IgMs belong to the same (III and II) lambda subgroups. By testing their antibody activity it was found by immunofluorescence and immunoblotting that only IgM2 reacts with an intermediate filament protein. Images Fig. 1 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Mendez, E; Osuna, C; Sanchez, A; Revilla, Y; Soriano, F; Montalban, C; Segui, J; Avila, J

1992-01-01

396

[Role of line immunoassay in the diagnosis of early HIV infection: a diagnostic case].  

PubMed

Combined p24 antigen-HIV antibody fourth-generation assays that identify most of the early HIV infections have been used extensively worldwide for several years. This poses challenges for the traditional algorithm of line immunoassay (LIA) confirmation. LIA tests are useful methods with their high specificity and their ability to differentiate HIV-1 from HIV-2, but they are reactive days after the fourth generation enzyme immunoassays. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. Serum sample of a patient sent to Ege University Hospital Clinical Virology Laboratory was repeatedly reactive with low signal/cutoff ratios with two different commercial fourth generation enzyme immunoassays (Architect HIV Ag/Ab Combo Reagent Kit, Abbott, Germany and Vidas HIV Duo Quick, Biomerieux, France). The sample was non-reactive with the LIA (INNO-LIA HIV I/II Score, Innogenetics, Belgium) and HIV RNA (RealTime HIV-I Amplification Reagent Kit, Abbott, USA) result was positive (4.1 x 10(5) copies/ml). With the presentation of this case, the role of LIA in the diagnosis of early HIV infection and its place in test algorithms were questioned. PMID:23971936

Soylar, Muhammed; Altu?lu, Imre; Sertöz, Rüçhan; Gökengin, Deniz

2013-07-01

397

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

SciTech Connect

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

Weis, J.K.

1988-01-01

398

The significance of IgM antinuclear antibody in rheumatoid arthritis and other connective tissue diseases  

Microsoft Academic Search

An investigation of the incidence of IgG and IgM antinuclear antibodies (ANA) in patients with connective tissue diseases showed that IgM ANA predominated in rheumatoid arthritis, whilst in systemic lupus erythematosus IgG antibodies were more common. Patients with other connective tissue diseases less frequently had antinuclear antibodies and there was little difference in the incidence of IgG and IgM antibodies.

M. C. Chellingworth; M. Salmon; D. L. Scott; P. A. Bacon

1984-01-01

399

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

400

A comparative trial of anti-glycoconjugate antibody assays: IgM antibodies to GM1  

Microsoft Academic Search

IgM class antibodies against the ganglioside GM1 have been found in a subgroup of patients with lower motor neuron syndromes and multifocal motor neuropathies (MMN). The pathogenic relevance of these antibodies is still unclear, but some MMN patients with IgM antibodies against GM1 seem to profit from immunosuppressive therapy. A reliable test for IgM antibodies against GMl may be useful

J. Zielasek; G. Ritter; S. Magi; H. P. Hartung; K. V. Toyka

1994-01-01

401

Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation.  

PubMed

We evaluated an automated chemiluminescence immunoassay (CLIA) developed for the measurement of urinary free deoxypyridinoline (DPD). The new DPD method by CLIA is based on the competition of DPD with particle-bound pyridinoline for a limited amount of monoclonal mouse anti-DPD antibody. Total imprecision (CV) was 3.2-9.0% at 30-270 nmol/L. Regression analysis of urinary DPD concentration (second morning-void) measured by CLIA (y) and enzyme immunoassay (EIA) for adult volunteers (n = 449) with and without bone disease revealed a best fit equation of: y = 1.08 +/- 0.03x - 1.15 +/- 0.98 nmol/L (r = 0.964, S(y/x) = 14 nmol/L). CLIA and EIA methods were correlated with HPLC measurement of urinary free DPD (r = 0.846 and 0.871, respectively). For healthy adults, the creatinine-normalized excretion of DPD (mean +/- SD) measured by CLIA for 61 men (4.1 +/- 1.2 micromol DPD/mol creatinine) and 76 premenopausal women (5.3 +/- 1.8 micromol DPD/mol creatinine) did not differ significantly (P >0.05) from DPD excretion measured by EIA, and both immunoassays showed a significant gender difference (P <0.001) in reference intervals. In a clinical trial, DPD excretion (micromol DPD/mol creatinine) measured by CLIA differed substantially from the reference population for 54 untreated pagetic (12.7 +/- 8.0 SD), 255 untreated osteoporotic (7.5 +/- 4.1), 21 osteomalacic (12.4 +/- 8.5), 17 primary hyperparathyroid (9.4 +/- 4.4), and 14 secondary hyperparathyroid (9.2 +/- 5.1) patients. Clinical sensitivities of the CLIA and EIA methods range from 38% to 80% in bone disorders and limit the use of the DPD measurement in disease detection. DPD excretion after pamidronate treatment in a subgroup of the pagetic patients fell dramatically as assessed by CLIA or EIA. We conclude that the automated CLIA method for DPD is a convenient and reliable method that may aid in the evaluation and management of bone disease and is applicable to high volume testing in the routine clinical laboratory. PMID:9761245

Rosano, T G; Peaston, R T; Bone, H G; Woitge, H W; Francis, R M; Seibel, M J

1998-10-01

402

Antigen-driven Induction of Polyreactive IgM during Intracellular Bacterial Infection  

PubMed Central

Polyreactivity is well known as a property of natural IgM produced by B-1 cells. We demonstrate that polyreactive IgM is also generated during infection of mice with Ehrlichia muris, a tick-borne intracellular bacterial pathogen. The polyreactive IgM bound self and foreign antigens, including single- and double-stranded DNA, insulin, thyroglobulin, lipopolysaccharide, influenza virus, and Borrelia burgdorferi. Production of polyreactive IgM during infection was antigen-driven, not due to polyclonal B cell activation, as the majority of polyreactive IgM recognized ehrlichial antigen(s), including an immunodominant outer membrane protein-19 (OMP-19). Monoclonal polyreactive IgM derived from T cell-independent spleen plasmablasts, which was germline-encoded, also bound cytoplasmic and nuclear antigens in HEp-2 cells. Polyreactive IgM protected immunocompromised mice against lethal bacterial challenge infection. Serum from human ehrlichiosis patients also contained poly- and self-reactive IgM. We propose that polyreactivity increases IgM efficacy during infection, but may also exacerbate or mollify the response to foreign and self antigens.

Jones, Derek D.; DeIulio, Gregory A.; Winslow, Gary M.

2012-01-01

403

Immunoassays with protein misfolding cycle amplification: a platform for ultrasensitive detection of antigen.  

PubMed

Protein misfolding cycle amplification (PMCA), a novel technology on amplifying cyclically misfolded proteins in vitro, is conceptually analogous to DNA amplification by polymerase chain reaction (PCR) and has tremendous implications for the researches and diagnosis. Here we first introduce the protein amplification technology into the classic immunoassay and develop a PMCA-based immunoassay (immuno-PMCA) for highly sensitive detection of antigen. This method takes advantage of sandwich binding of two affinity aptamers for increased specificity, magnetic nanoparticles for fast magnetic separation, PMCA for signal amplification, and conjugated polyelectrolytes for visual detection, allowing the detection limit of antigen by colorimetry down to femtomolar level with a wide linear range from 10 to 10(4) fM. More importantly, no specialized facilities or enzymes are needed either in the amplification reaction or the evaluation of results, which indicates its great potential application in immunological research and clinical diagnostics. PMID:22888831

Zhou, Peng; Luo, Qingying; Lin, Yi; Chen, Lu; Li, Sha; Zhou, Guohua; Ji, Xinghu; He, Zhike

2012-08-23

404

Immunoassay using microfluid filters constructed by deep X-ray lithography.  

PubMed

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay. PMID:18071245

Matsui, Katsuhiro; Kawaji, Isao; Utsumi, Yuichi; Ukita, Yoshiaki; Asano, Toshifumi; Takeo, Masahiro; Kato, Dai-ichiro; Negoro, Seiji

2007-12-07

405

The ProtoChip™ immunoassay biochip  

Microsoft Academic Search

ProtoPharm makes use of the ProtoChip™, a high-density immunoassay system, to construct epitope “fingerprints” of complex protein mixtures. Unlike all other protein biochip methods, the ProtoChip does not require the immobilization of proteins or antibodies on a solid support nor does the ProtoChip require the purification of large numbers of proteins. The ProtoChip may be used to profile complex protein

David P Dumas

2002-01-01

406

Immunoassays for drug screening in urine  

Microsoft Academic Search

Immunoassays are presently used worldwide for the rapid screening of drugs. Despite the fact that they are a highly valuable\\u000a tool for the testing of legal and illicit drugs, there is a real risk of false-positive and false-negative findings and many\\u000a pitfalls must be taken into account when these tests are used in an uncritical manner and without valid confirmation

Harald Schütz; Alexandre Paine; Freidoon Erdmann; Günter Weiler; Marcel A. Verhoff

2006-01-01

407

Development of SCA (Trade Name) Protein with Murine IgM Specificity, Phase 1 Final Report.  

National Technical Information Service (NTIS)

The project involves development of a single-chain antigen-binding (SCA) protein with specific affinity for mouse IgM. A protein which specifically and reversibly binds murine IgM has application in the large-scale purification and separation of murine Ig...

D. R. Filpula

1990-01-01

408

Development and preliminary validation of an antibody filtration-assisted single-dilution chemiluminometric immunoassay for potency testing of Piscirickettsia salmonis vaccines.  

PubMed

Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples. PMID:23040097

Wilda, Maximiliano; Lavoria, María Ángeles; Giráldez, Adrián; Franco-Mahecha, Olga Lucía; Mansilla, Florencia; Érguiz, Matías; Iglesias, Marcela Elvira; Capozzo, Alejandra Victoria

2012-10-02

409

Natural IgM in immune equilibrium and harnessing their therapeutic potential  

PubMed Central

Natural IgM antibodies are the constitutively secreted products of B1 cells (CD5+ in mice and CD20+CD27+CD43+ CD70? in humans) that have important and diverse roles in health and disease. Whereas the role of natural IgM as first line of defense for protection against invading microbes has been extensively investigated, more recent reports have highlighted their potential roles in the maintenance of tissue homeostasis via clearance of apoptotic and altered cells through complement-dependent mechanisms, inhibition of inflammation, removal of misfolded proteins, and regulation of pathogenic auto-reactive IgG antibodies and autoantibody-producing B cells. These observations have provided the theoretical underpinnings for efforts that currently seek to harness the untapped therapeutic potential of natural IgM either by boosting in vivo natural IgM production or via therapeutic infusions of monoclonal and polyclonal IgM preparations.

Kaveri, Srini V.; Silverman, Gregg J.; Bayry, Jagadeesh

2011-01-01

410

Prevalence of Legionella-specific IgG and IgM antibody in a dental clinic population.  

PubMed

This study was undertaken to determine the frequency of Legionella infection in a dental clinic setting. Serum samples from 270 dental clinic personnel were evaluated using an enzyme-linked immunosorbent assay to detect Legionella-specific IgM and IgG antibodies. The pooled-species whole-cell-antigen preparation used in these assays was derived from six Legionella pneumophila strains and one strain each from Legionella bozemanii and Legionella micdadei. Significant levels of IgG and IgM antibodies were found in 20% and 16%, respectively, of the samples. This compares with 8% and 10%, respectively, for a randomly selected non-clinical group from the region (P less than 0.005). Samples from clinic personnel with significant IgG titers (greater than 1:128) were also evaluated for activity to each of the eight single-species antigens, with the following results: L. pneumophila, 45% (combined six strains); L. micdadei, 37%; and L. bozemanii, 18%. Comparing individuals' "years spent in the clinic environment" with the incidence of significant antibody levels strongly suggests that the risk of Legionella infection increases proportionately with increased clinic exposure time (P less than 0.05). Analysis of these data implies that Legionella may be present in the dental clinic environment, thus creating an increased risk for clinical personnel or patients. PMID:3865949

Fotos, P G; Westfall, H N; Snyder, I S; Miller, R W; Mutchler, B M

1985-12-01

411

Saliva IgM and IgA are a sensitive indicator of the humoral immune response to Escherichia coli O157 lipopolysaccharide in children with enteropathic hemolytic uremic syndrome.  

PubMed

Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS). Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E. coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA. Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values. Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E. coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected. These results closely mirrored the results obtained with paired serum samples. In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies. The specificity of the ELISA was confirmed by western blotting. In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included. The specificity was 98% for IgM and 100% for IgA. Children with E. coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva. Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E. coli O157 infection in patients with enteropathic HUS. PMID:12149511

Ludwig, Kerstin; Grabhorn, Enke; Bitzan, Martin; Bobrowski, Christoph; Kemper, Markus J; Sobottka, Ingo; Laufs, Rainer; Karch, Helge; Müller-Wiefel, Dirk E

2002-08-01

412

Assay of Chlamydia pneumoniae-Specific IgM Antibodies by ELISA Method---Reduction of Non-Specific Reaction and Resetting of Serological Criteria by Measuring IgM Antibodies  

Microsoft Academic Search

SUMMARY: In the present study, we tried to reduce the non-specific reactions for measuring anti-Chlamydia pneumoniae IgM antibodies by the ELISA kit of HITAZYME C. pneumoniae Ab-IgM (HITAZYME IgM) by using a new absorbent. We also tried to reset the IgM cut-off index (ID) of HITAZYME IgM by testing serum samples from healthy children and healthy adults with no respiratory

Toshio Kishimoto; Shuji Ando; Kei Numazaki; Kazunobu Ouchi; Tsutomu Yamazaki; Chikara Nakahama

413

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

414

Development of monoclonal antibody-based immunoassays to the N-methylcarbamate pesticide carbofuran.  

PubMed

To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently characterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterologous assay haptens. Depending on the immunoreagent combination and assay format, competitive assays with I(50) values in the 1.2-10.2 nM (0.27-2.27 ng/mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFNH, coupled either to horseradish peroxidase or to ovalbumin, was used to develop a direct and an indirect enzyme-linked immunosorbent assay, respectively. Optimized immunoassays displayed very similar analytical characteristics, with an I(50) value around 0.7 ng/mL and a limit of detection around 0.08 ng/mL. Both immunoassays were able to tolerate the presence of methanol up to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pesticides were not recognized by the MAb. These immunoassays should reasonably allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practical importance. PMID:10794653

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

1999-06-01

415

High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction.  

PubMed Central

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays. Images

Hendrickson, E R; Truby, T M; Joerger, R D; Majarian, W R; Ebersole, R C

1995-01-01

416

Validation of a new homogeneous immunoassay for the detection of carisoprodol in urine.  

PubMed

The objective of this project was to validate a new high-throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of carisoprodol in human urine. Carisoprodol (Soma(®)) and meprobamate are widely prescribed as musculoskeletal pain relief drugs and are listed as one of the 10 most frequently identified drugs associated with DUI cases. Carisoprodol has a short elimination half-life of 1-3 h; however, its major active metabolite, meprobamate, has a longer elimination half-life of 6-17 h. As a result, it is important for an immunoassay to cross-react with both compounds. The advantage of this new assay is that cutoff concentrations can be adjusted between 100 and 500 ng/mL. The reportable range was 25 to 1000 ng/mL for carisoprodol and 50 to 10,000 ng/mL for meprobamate. The intraday coefficient of variation (% CV) for the semi-quantitative assay was less than 1%. The homogeneous assay was validated with a total of 86 urine samples previously analyzed by liquid chromatography-tandem mass spectrometry with carisoprodol concentrations ranging from 50 to 10,000 ng/mL. The accuracy was found to be 100% when immunoassay cutoff concentrations of carisoprodol and meprobamate were set at 100 and 1000 ng/mL, respectively. PMID:21396230

Wang, Guohong; Huynh, Kim; Barhate, Rekha; Rodrigues, Warren; Moore, Christine; Coulter, Cynthia; Vincent, Michael; Soares, James

2011-03-01

417

Sensitive detection of food-borne pathogen Salmonella by modified PAN fibers-immunoassay.  

PubMed

Sensitive and rapid detection of Salmonella is a key to the prevention and identification of problems associated with human health and safety. Enzyme Linked Immunosorbent Assays (ELISAs) are popular and widely implemented technique to detect pathogenic bacteria in routine analysis but a typical ELISA yields a sensitivity of 10(6)-10(7)cfu/mL. The present study consecrates on the applicability of surface modified polyacrylonitrile (PAN) fibers as a novel matrix of immunoassay for the detection of Salmonella typhimurium in a sandwich ELISA format. Affinity purified antibody against Salmonella common structural antigen (CSA-1-Ab) was immobilized on modified PAN (mPAN) fibers using covalent immobilization via amine-glutaraldehyde chemistry and inactivated S. typhimurium were captured from various samples and detected colorimetrically using peroxidase-labelled common structural antibody (CSA-1-Ab-HRP) against Salmonella. The performance of the developed immunoassay was compared with commercially available immunomagnetic microbeads (Dynabeads(®) anti-Salmonella), polystyrene (PS) microtitre plate and glutaraldehyde activated PS plate. Limit of detection (LOD) was found to be 10, 10(5), 10(6) and 10(7)cells/mL of bacteria for mPAN, Dynabeads(®), glu-plate and PS plate respectively without any pre-enrichment step. The assay was specific for the targeted bacteria when investigated with other cross-reactant food and water-borne pathogens. The developed immunoassay offered undisputed advantages of being simple, sensitive and specific for the detection of S. typhimurium. PMID:23500375

Chattopadhyay, Sruti; Kaur, Avneet; Jain, Swati; Singh, Harpal

2013-02-11

418

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. ...

419

Accelerated development of IgG autoantibodies and autoimmune disease in the absence of secreted IgM  

Microsoft Academic Search

Individuals with systemic lupus erythematosus and rheumatoid arthritis are characterized by the presence of high levels of circulating IgM and IgG autoantibodies. Although IgG autoantibodies often are pathogenic, the role of IgM autoantibodies in autoimmune disease is not clear. Using mice that are unable to secrete IgM but are able to express surface IgM and IgD and to secrete other

Marianne Boes; Tara Schmidt; Kathrin Linkemann; Britte C. Beaudette; Ann Marshak-Rothstein; Jianzhu Chen

2000-01-01

420

Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva  

Microsoft Academic Search

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added

L Anfossi; C Tozzi; C Giovannoli; C Baggiani; G Giraudi

2002-01-01

421

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

PubMed Central

A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e. g. insecticides and chemical nerve agents through directly detecting OP phosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly-efficient magnetic separation with ultra-sensitive square wave voltammetry (SWV) analysis using quantum dots (QDs) as labels. A pair of antibodies were utilized to achieve the specific recognition of OP-AChE that was prepared using paraoxon as an OP model agent. Anti-phosphoserine polyclonal antibodies (Ab1) were anchored on amorphous magnetic particles (MPs) preferably chosen to capture OP-AChE from the sample matrixes through binding their phosphoserine moieties that were exposed through unfolding the protein adducts, which was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, anti-human AChE monoclonal antibodies (Ab2) were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy (TEM). The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes (SPEs). Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3 – 300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promises as a simple, selective, sensitive, and field analysis tool for the effective bio-monitoring and diagnosis of potential exposures to nerve agents and pesticides.

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

2009-01-01

422

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

SciTech Connect

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

Wang, H