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1

Detection of IgM antibodies against Chlamydia trachomatis by enzyme linked fluorescence immunoassay.  

PubMed Central

A simple, sensitive enzyme linked fluorescence immunoassay has been developed to detect IgM antibodies against Chlamydia trachomatis. Reticulate bodies and elementary bodies from C trachomatis L2/434 Bu strain were isolated and used as antigens in the assay. Of 113 serum samples obtained from infants with pneumonia, 27 (23.9%) had IgM antibodies to C trachomatis L2 reticulate bodies and nine (8.0%) had IgM antibodies to C trachomatis L2 elementary bodies (titre greater than or equal to 1/500). Specific IgM antibodies were not detected in 20 control serum samples obtained from healthy adults and children. The possible use of enzyme linked fluorescence assay to determine IgM antibodies in the serodiagnosis of C trachomatis infection is discussed. PMID:3894429

Numazaki, K; Chiba, S; Yamanaka, T; Moroboshi, T; Aoki, K; Nakao, T

1985-01-01

2

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.  

PubMed Central

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

Crouch, C F

1995-01-01

3

Enzyme immunoassay for methamphetamine.  

PubMed

A competitive enzyme immunoassay for methamphetamine with alkaline phosphatase labeled methamphetamine, Sepharose-antibody and p-nitrophenylphosphate as substrate was developed. The anti-methamphetamine antisera produced in rabbits by immunization with N-(4-aminobutyl) methamphetamine-BSA conjugate were specific for methamphetamine and showed low cross-reactivities with p-OH methamphetamine and amphetamine (metabolites of methamphetamine). The range of methamphetamine measurable by the enzyme immunoassay was 1 to 300 ng/tube. According to the assay, methamphetamine could be detected from urine and extract of hair. PMID:6343585

Aoki, K; Kuroiwa, Y

1983-01-01

4

Gold bands as a suitable surface for enzyme immunoassays.  

PubMed

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

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

2002-09-01

5

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

6

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

7

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

8

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

9

Multiplex Detection of IgM and IgG Class Antibodies to Toxoplasma gondii, Rubella Virus, and Cytomegalovirus Using a Novel Multiplex Flow Immunoassay ?  

PubMed Central

The goal of this study was to evaluate the BioPlex 2200 Toxoplasma, rubella, and cytomegalovirus (CMV) (ToRC) IgG and IgM multiplex immunoassays (Bio-Rad Laboratories, Hercules, CA) and compare the results to those of conventional testing by enzyme immunoassay (EIA) and enzyme-linked fluorescent assay (ELFA). Serum specimens (n = 600) submitted for routine ToRC IgG and IgM testing by EIA (SeraQuest, Doral, FL; Diamedix, Miami, FL) or ELFA (Vidas; bioMérieux, Durham, NC) were also tested by the BioPlex ToRC multiplex immunoassays. Samples showing discordant results were retested by both methods, with further discrepancies being arbitrated by a third assay. Following repeat testing, the BioPlex Toxoplasma, rubella, and CMV IgG assays demonstrated agreements of 98.7 (592/600 specimens), 93.3 (560/600 specimens), and 98.3% (590/600 specimens), respectively, while the ToRC IgM assays yielded agreements of 91.2 (547/600 specimens), 87.3 (524/600 specimens), and 95.2% (571/600 specimens), respectively. The BioPlex ToRC IgG assays provided results comparable to EIA/ELFA results, with kappa coefficients showing near-perfect agreement for the Toxoplasma (? = 0.94) and CMV (? = 0.97) IgG assays and substantial agreement for the rubella IgG assay (? = 0.66). The BioPlex ToRC IgM assays showed lower specificity with only slight agreement for Toxoplasma IgM (? = 0.07), poor agreement for rubella IgM (? = ?0.03), and moderate agreement for CMV IgM (? = 0.55). Both the BioPlex IgG and IgM assays reduced turnaround time (1.7 h versus 5.5 h by EIA/ELFA for 100 specimens) and eliminated the necessity to manually pipette or aliquot specimens prior to testing. PMID:20861325

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

2010-01-01

10

Multiplex detection of IgM and IgG class antibodies to Toxoplasma gondii, rubella virus, and cytomegalovirus using a novel multiplex flow immunoassay.  

PubMed

The goal of this study was to evaluate the BioPlex 2200 Toxoplasma, rubella, and cytomegalovirus (CMV) (ToRC) IgG and IgM multiplex immunoassays (Bio-Rad Laboratories, Hercules, CA) and compare the results to those of conventional testing by enzyme immunoassay (EIA) and enzyme-linked fluorescent assay (ELFA). Serum specimens (n = 600) submitted for routine ToRC IgG and IgM testing by EIA (SeraQuest, Doral, FL; Diamedix, Miami, FL) or ELFA (Vidas; bioMérieux, Durham, NC) were also tested by the BioPlex ToRC multiplex immunoassays. Samples showing discordant results were retested by both methods, with further discrepancies being arbitrated by a third assay. Following repeat testing, the BioPlex Toxoplasma, rubella, and CMV IgG assays demonstrated agreements of 98.7 (592/600 specimens), 93.3 (560/600 specimens), and 98.3% (590/600 specimens), respectively, while the ToRC IgM assays yielded agreements of 91.2 (547/600 specimens), 87.3 (524/600 specimens), and 95.2% (571/600 specimens), respectively. The BioPlex ToRC IgG assays provided results comparable to EIA/ELFA results, with kappa coefficients showing near-perfect agreement for the Toxoplasma (? = 0.94) and CMV (? = 0.97) IgG assays and substantial agreement for the rubella IgG assay (? = 0.66). The BioPlex ToRC IgM assays showed lower specificity with only slight agreement for Toxoplasma IgM (? = 0.07), poor agreement for rubella IgM (? = -0.03), and moderate agreement for CMV IgM (? = 0.55). Both the BioPlex IgG and IgM assays reduced turnaround time (1.7 h versus 5.5 h by EIA/ELFA for 100 specimens) and eliminated the necessity to manually pipette or aliquot specimens prior to testing. PMID:20861325

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

2010-11-01

11

Enzyme immunoassays with special reference to ELISA techniques  

Microsoft Academic Search

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.

A Voller; A Bartlett; D E Bidwell

1978-01-01

12

[Detection of food additives by enzyme immunoassay].  

PubMed

The analysis of synthesized food additives is generally performed by chromatography or spectrophotometry. However, the analytical procedures for natural food additives have been little reported so far because they are difficult to analyse chemically. We have attempted to apply enzyme immunoassay (EIA) to the analysis of natural food additives. Hen egg white lysozyme, as a food preservative, was determined by the competitive EIA, using mouse anti-HEL ascites. Carminic acid (CA), which is the main component of cochineal color, was determined by the competitive EIA, using monoclonal anti-CA antibody. Phycocyanin, which is the main component of spirulina color, was determined by the avidin-biotin sandwich EIA, using double monoclonal anti-phycocyanin antibodies. PMID:7474399

Yoshida, A; Takagaki, Y

1995-09-01

13

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

PubMed

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

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

2011-05-01

14

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

EPA Science Inventory

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

15

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

PubMed

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

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

2014-10-01

16

Measuring multiple hormones from a single water sample using enzyme immunoassays  

E-print Network

Measuring multiple hormones from a single water sample using enzyme immunoassays Celeste E. Kidd F2a 11-Ketotestosterone Astatotilapia burtoni Enzyme immunoassay EIA Teleost Cichlid Steroid radioimmuno- assay (RIA) or enzyme immunoassay (EIA) is becoming widely used in teleost research. Commercial

Hofmann, Hans A.

17

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...

18

Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi.  

PubMed Central

A commercial enzyme immunoassay kit called the Dip-S-Ticks (DS) for the detection of total immunoglobulin (Ig) G and IgM human antibodies to Rickettsia typhi was evaluated. In tests with 340 serum samples from patients with diagnosed cases of rickettsial diseases, patients suffering from other febrile illnesses, and normal subjects, the DS compared favorably with the standard indirect fluorescent-antibody (IFA) test. At IFA cutoff titers of > or = 1.64 and > or = 1:128, the DS showed sensitivities of 88.2 and 91.4% and specificities of 91.8 and 87.7%, respectively. The DS test correlated significantly with both the IFA IgG (r = 0.84, P < 0.0005) and IgM (r = 0.63, P < 0.0005) titers. Only 80% of IgG and 82% of IgM IFA readings determined by two technicians were within one dilution, while the DS was more reliable, with 100% within one dot. The rapidity, reliability, and simplicity of the DS suggest that it is a suitable test for use in clinical laboratories unable to perform the IFA test. PMID:7664182

Kelly, D J; Chan, C T; Paxton, H; Thompson, K; Howard, R; Dasch, G A

1995-01-01

19

Alternative immunoassays  

SciTech Connect

This book contains 13 chapters. Some of the chapter titles are: Uses of immunoassay; Particle immunoassays; Immunoradiometric assays; Enzyme immunoassays; and Current concepts and future developments.

Collins, W.P.

1985-01-01

20

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

EPA Science Inventory

Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic ...

21

Enzyme immunoassay validation detection of cocaine in for qualitative sweat  

Microsoft Academic Search

A solid-phase enzyme immunoassay (EIA) involving micro- titer plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek?' sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE)

VINA SPIEHL ER; JOHN FAY; ROBERT FOGERSON; DON SCHOENDORFER; R. SAM NIEDBALA

22

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

23

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex  

NASA Astrophysics Data System (ADS)

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

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

1999-04-01

24

Development and evaluation of a Sarcocystis neurona-specific IgM capture enzyme-linked immunosorbent assay.  

PubMed

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P < .0001). For horses inoculated with lower doses of S neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM. PMID:16594589

Murphy, J E; Marsh, A E; Reed, S M; Meadows, C; Bolten, K; Saville, W J A

2006-01-01

25

Dot-blot enzyme immunoassay for the detection of bovine viral Dot-blot enzyme immunoassay for the detection of bovine viral diarrhea virus antibodies diarrhea virus antibodies  

Microsoft Academic Search

ABSTRACT Dot-blot enzyme immunoassay (DB-EIA) was utilized for the detection of bovine viral diarrhea (BVD) Dot-blot enzyme immunoassay (DB-EIA) was utilized for the detection of bovine viral diarrhea (BVD) antibodies in infected cattle. In this assay whole particles of the NADL strain of BVD virus were used as antibodies in infected cattle. In this assay whole particles of the NADL

Farhid Hemmatzadeh; Farhad Amini

26

Whole blood cyclosporin monitoring in liver and heart transplant patients: evaluation of the specificity of a fluorescence polarization immunoassay and an enzyme-multiplied immunoassay technique  

Microsoft Academic Search

The specificity of two cyclosporin immunoassays were evaluated. Eleven patients were followed for the first four weeks after heart (n = 3) or liver (n = 8) transplantation. Cyclosporin A (CsA) monitoring was performed concomitantly by a monoclonal fluorescence polarization immunoassay (mFPIA) and enzyme-multiplied immunoassay technique (EMIT®) during this period. For several patients, cyclosporin monitoring was also performed by high

Béatrice Gulbis; Jack Van der Heijden; Harry van As; Philippe Thiry

1997-01-01

27

Enzyme immunoassay of thyroxin with a centrifugal analyzer  

SciTech Connect

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

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

1982-01-01

28

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay.  

PubMed

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change. PMID:3532208

Hiroshima, O; Hayashi, H; Ito, S; Hayaishi, O

1986-07-01

29

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

E-print Network

Comparison of a Time-Resolved Fluorescence Immunoassay and an Enzyme-Linked Immunosorbent Assay of California, Davis, California 95616 Immunoassays for atrazine based on a time-resolved fluorescent label immunoassay (TRFIA) was based on a polyclonal antibody and a europium label, whereas the enzyme

Hammock, Bruce D.

30

Immunoassay  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

31

EnzymeImmunoassay, KineticMicroparticle Immunoassay,Radioimmunoassay, and Fluorescence PolarizationImmunoassay Comparedfor Drugs-of-Abuse Screening  

Microsoft Academic Search

The newestformulation ofthe Syva EMIT assayfor drugs of abuse, EMIT II, and a new immunoassay, OnUne (Roche), utilizing the kinetic interaction of microparticles in solution(KIMS)methodology, RIA tests,and TDx fluo- rescence polarization immunoassay (FPIA) procedures were compared for marijuana,cocaine,opiates,and bar- biturates.BothEMIT II and OnLineimmunoassays were performedwitha Hitachi717 analyzer.Calibration curves, the degree of separationbetween negativeand cutoff calibrators, precision,likelihood ofcarryoverfrompositive to negativesamples,andoverallease andspeedofanal-

David A. Armbruster; Robert H. Schwarzhoff; Edward C. Hubster; Monica K. Liserio

32

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

33

Rapid dioxin screening of milk and water by enzyme immunoassay  

SciTech Connect

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

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

1995-12-01

34

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

35

Enzyme conversion immunoassay for determining total homocysteine in plasma or serum  

Microsoft Academic Search

A rapid and precise immunoassay for quantification of total homocysteine in blood samples is presented. The method avoids the use of radioisotopes and chromato- graphic separations and relies on enzymatic conversion of homocysteine to S-adenosyl-L-homocysteine, fol- lowed by quantification of S-adenosyl-L-homocysteine by an enzyme-linked immunoassay in microtiter format. The within- and between-assay imprecision is <6% and 8%, respectively, and results

Frank Frantzen; Arne Ludvig Faaren; Ingrid Alfheim; Arne Kristian Nordhei

36

Immunoassays  

Microsoft Academic Search

Immunoassays are used in basic biological research to investigate the physiological and possible pathological role of a wide range of biologically active substances including cyclic nucleotides, prostaglandins, leukotrienes, growth factors and cytokines [1]. Such research often leads to the identification of new potential targets for therapeutic agents.The assays are also used in the pharmaceutical industry in many aspects of the

Michael J. O'Sullivan

37

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

38

Determination of tumor marker CA125 by capillary electrophoretic enzyme immunoassay with electrochemical detection  

Microsoft Academic Search

A novel capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) was developed for a tumor marker cancer antigen 125 (CA125). In this method, after the noncompetitive enzyme immunoreaction, the free enzyme (horseradish peroxidase)-labeled anti-CA125 antibody (Ab?) and the bound enzyme-labeled complex (Ag–Ab?) were separated in a separation capillary and then catalyzed the enzyme substrate (3,3,5,5-tetramethyl-benzidine dihydrochloride, TMB(Red)) and H2O2 in

Zhihui He; Ning Gao; Wenrui Jin

2003-01-01

39

Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay  

Microsoft Academic Search

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days

ATHANASIOS MAKRISTATHIS; EVA PASCHING; KURT SCHUTZE; MARGIT WIMMER; MANFRED L. ROTTER; ALEXANDER M. HIRSCHL

1998-01-01

40

Assessment of Platelia Aspergillus enzyme immunoassay for the diagnosis of invasive aspergillosis  

Microsoft Academic Search

Background and Purpose: This study investigated the diagnostic value of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen in patients at risk of invasive aspergillosis (IA), and its association with clinical course and outcome. Methods: A total of 304 blood samples were collected from 189 patients at risk of IA during a 1-year period at a tertiary referral center.

Chih-Cheng Lai; Hsiao-Leng Hsu; Li-Na Lee; Po-Ren Hsueh

41

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...

42

Evaluation of a new enzyme immunoassay for detecting Helicobacter pylori in feces: a prospective pilot study  

Microsoft Academic Search

OBJECTIVE: There is an increasing interest in noninvasive tests for detecting Helicobacter pylori (H. pylori) infection. Unlike serological and urea breath tests, the possibility of searching for H. pylori in feces has been scarcely investigated. The aim of this prospective pilot study was to evaluate the usefulness of a new enzyme immunoassay for detecting H. pylori antigens in feces, as

Lucio Trevisani; Sergio Sartori; Fabrizio Galvani; Maria Rita Rossi; Marco Ruina; Carlo Chiamenti; Michele Caselli

1999-01-01

43

Enzyme-linked sandwich immunoassay for insulin using laser fluorimetric detection.  

PubMed Central

Human serum samples are assayed for insulin by an enzyme-linked sandwich immunoassay. Horseradish peroxidase is used as an enzyme label for antibody, and enzyme activity is measured by means of the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325-nm line of a continuous-wave helium/cadmium ion laser on line with reverse-phase high-pressure liquid chromatography. The incubation period is 90 min and the limit of detection of insulin is 30 pM, corresponding to 5 microunits/ml. This method correlates highly with radioimmunoassay, with coefficient of correlation r = 0.95. PMID:7015349

Lidofsky, S D; Hinsberg, W D; Zare, R N

1981-01-01

44

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

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

45

A microplate enzyme-immunoassay for toxoplasma antibody  

Microsoft Academic Search

A new test for the detection and measurement of toxoplasma antibody is described. Test sera are reacted with antigen-sensitized wells in micro-haemagglutination plates. Any attached antibody is shown by the addition of an enzyme-labelled antiglobulin followed by assay of the enzyme reaction with its substrate. The test is easy to carry out on a large scale, and there is a

A Voller; D E Bidwell; A Bartlett; D G Fleck; M Perkins; B Oladehin

1976-01-01

46

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

Microsoft Academic Search

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

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

2007-01-01

47

Evaluation of the reliability of 6 current anti-HIV1\\/HIV2 enzyme immunoassays  

Microsoft Academic Search

The sensitivity for early detection of HIV antibodies and specificity of 6 anti-HIV-1\\/HIV-2 screening enzyme immunoassays (ELISAs) currently on the market were investigated by testing a panel of 249 well-characterized serum samples. The panel included sera from AIDS patients or children with congenital HIV infection, high-risk individuals and patients with conditions unrelated to AIDS. ‘Tricky’ sera (repeatedly positive results by

Bernard Weber; Mahin Moshtaghi-Boronjeni; Michael Brunner; Wolfgang Preiser; Markus Breiner; Hans Wilhelm Doerr

1995-01-01

48

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

49

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

50

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

51

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

NASA Astrophysics Data System (ADS)

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

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

1982-05-01

52

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

PubMed Central

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

Kurstak, Edouard

1985-01-01

53

A multi?residue enzyme immunoassay for screening illegally used ??agonists  

Microsoft Academic Search

A multi?residue screening test was developed which provides reliable detection of eight ß?agonists in urine, based on an enzyme immunoassay with peroxidase and an antibody raised against clenbuterol?diazo?BSA. Antibodies were compared after continuous immunization. The antibody obtained after the 12th immunization showed the best sensitivity, procedural blanks of urine samples and coefficients of variation, using analysis of spiked urine samples.

Inge Dürsch; Heinrich H. D. Meyer

1992-01-01

54

Surface electrochemical enzyme immunoassay for the highly sensitive measurement of B-type natriureric peptide  

Microsoft Academic Search

B-type natriuretic peptide (BNP), a peptide hormone, was determined by electrochemical enzyme immunoassay through the following procedure. First, a certain concentration of sample BNP was added to the solution containing anti-BNP antibody modified with acetylcholinesterase (AChE) to undergo the immunological reaction. Then BNP-modified gold particles were added into the BNP\\/AChE-labeled anti-BNP antibody solution so that the unreacted AChE-labeled anti-BNP antibody

Hiroaki Matsuura; Yukari Sato; Osamu Niwa; Fumio Mizutani

2005-01-01

55

Typing of Human Astroviruses from Clinical Isolates by Enzyme Immunoassay and Nucleotide Sequencing  

Microsoft Academic Search

A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus- positive specimens from nine collections from seven countries. Six of the seven known astrovirus types were detected in the collections, with HAstV-1 predominating in all collections except for one from the United Kingdom. Selected specimens were analyzed further by reverse transcriptase PCR and nucleotide sequencing

JACQUELINE S. NOEL; TERRY W. LEE; JOHN B. KURTZ; ROGER I. GLASS

1995-01-01

56

Solidphase enzyme-immunoassay for detection of hepatitis B surface antigen  

Microsoft Academic Search

The preliminary results of a solid-phase enzyme-immunoassay (EIA) for the detection of hepatitis B surface antigen (HBsAg) are presented. This method has been compared with the solid-phase radioimmunoassay (RIA) for HBsAg in dilution series of four HBsAg positive sera four national reference panels (The Laboratory Panel of the Central Laboratory of the Blood Transfusion Service of the Netherlands Red Cross,

G Wolters; L Kuijpers; J Kacaki; A Schuurs

1976-01-01

57

Quantitative determination of nuclear estrogen receptors by an enzyme immunoassay: applicability and caveats.  

PubMed

A method is presented with which approx. 95% of nuclear estrogen receptors appear to be extracted from MCF-7 cells. Since both nuclear isolation and nuclear estrogen receptor extraction take place in a single test tube with only vortex mixing, loss of nuclear material is minimized. The amount of nuclear estrogen receptors in the nuclear extract was determined by direct [3H]estradiol labeling of monolayer cultures and with a commercially available estrogen receptor immunoassay (ER-EIA) kit. Since the ER-EIA kit was designed and calibrated for quantitative determination of cytosolic estrogen receptor isolated in low ionic strength buffer, the applicability of the ER-EIA to quantitative determination of estrogen receptor content in high ionic strength nuclear extraction buffer was tested. A linear relationship exists between the amount of nuclear estrogen receptor detected by the immunoassay, the amount of receptor present in serial dilutions of the nuclear extract and the amount of nuclear estrogen receptor detected in cells by [3H]estradiol labeling of monolayer cultures, the absolute amount of nuclear estrogen receptors determined by the immunoassay consistently exceeded the amount of receptor detected by [3H]estradiol labeling. The possibility that the enzyme immunoassay must be properly calibrated for the specific conditions of the nuclear estrogen receptor assay is discussed. PMID:3050279

Kral, L G; Doherty, L M; Brooks, S C

1988-10-01

58

Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF)  

SciTech Connect

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using /sup 125/I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.

Caruelle, D.; Grassi, J.; Courty, J.; Groux-Muscatelli, B.; Pradelles, P.; Barritault, D.; Caruelle, J.P.

1988-09-01

59

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.

60

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

PubMed

Enzymatic signal amplification based on fluorogenic substrates is commonly used for immunoassays; however, when transitioning these assays to a digital format in water-in-mineral oil emulsions, such amplification methods have been limited by the leakage of small reporting fluorescent probes. In the present study, we used a microfluidic system to study leakage from aqueous droplets in a controlled manner and confirmed that the leakage of fluorescent resorufin derivatives is mostly due to the presence of the lipophilic surfactant Span80, which is commonly used to preserve emulsion stability. This leakage can be overcome by the addition of specific sugars that most strongly interfered with the surfactants ability to form micelles in water. The application of the microfluidic system to the quantitative analysis of droplets and the implementation of the described sugar additives would allow for alternatives to fluorinated surfactant-based platforms and improve the signal fidelity in enzyme immunoassays implemented through multiphase microfluidics. PMID:24870310

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

2014-06-17

61

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

PubMed

Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3515886

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

1986-01-01

62

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

63

Rapid and simple method for detecting Salmonella in chicken feces using polymyxin-cloth enzyme immunoassay  

Microsoft Academic Search

The presence of three strains ofSalmonellacells in chicken feces was detected by polymyxin-cloth enzyme immunoassay. A cotton swab-full ofSalmonella-free chicken feces (containing an aerobic count of about 3×107cfu) was mixed with a small number ofSalmonella typhimurium(6 or 12 cfu),Salmonella enteritidis(6 or 15 cfu) orSalmonella hadar(5 or 10 cfu) in 5 ml of brain–heart infusion medium supplemented with 0.5% yeast extract,

S. Hayashi; H. Yamazaki

1996-01-01

64

Establishment and application of enzyme immunoassay for saliva cortisol in Taiwanese context.  

PubMed

Saliva steroid assay is an upcoming area of research, with much potential for growth and progress. Expensive, varying results with commercial kits and the disadvantages of radioimmunoassay have forced researchers to develop their own system of enzyme immunoassay (EIA). A modification from our established EIA system was used to develop a saliva cortisol (F) assay system. The system sensitivity (>90pg/mL) was checked by various experiments, including comparison of data with a commercial kit obtained from Salimetrics. The assay system was employed to investigate the saliva F level in a young Taiwanese population, and compared with the total and free serum levels of F. PMID:16827226

Chen, Han-Lin; Chen, Yung-Liang; Wu, Leang-Shin; Kaphle, Krishna; Lin, Jen-Hsou

2006-01-01

65

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

NASA Technical Reports Server (NTRS)

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

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

1987-01-01

66

Effect Of Temperature on the Sensitivity of Sandwich Enzyme Immunoassay with Fab'Horseradish Peroxidase Conjugate  

Microsoft Academic Search

Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher

Masayoshi Imagawa; Shinji Yoshitake; Seiichi Hashida; Eiji Ishikawa

1982-01-01

67

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

PubMed

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations. PMID:25766738

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

2015-05-01

68

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay  

Microsoft Academic Search

We present a new type of enzyme–antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily

Rongqin Ke; Wei Yang; Xiaohu Xia; Ye Xu; Qingge Li

2010-01-01

69

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. PMID:16467328

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

2006-01-01

70

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

SciTech Connect

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

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

1995-12-31

71

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

PubMed

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

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

2015-01-01

72

Evaluation of Performances of Three DNA Enzyme Immunoassays for Detection of Helicobacter pylori PCR Products from Biopsy Specimens  

Microsoft Academic Search

PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical labo- ratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three

LURDES MONTEIRO; FRANCIS MEGRAUD; Universitede Bordeaux

1997-01-01

73

Comparison of an enzyme immunoassay and latex agglutination test for detection of galactomannan in the diagnosis of invasive aspergillosis  

Microsoft Academic Search

Aspergillus antigenemia was followed up in 215 consecutively observed bone marrow transplant (BMT) patients over a period of two years, using both a latex agglutination test and a sandwich immunocapture enzyme immunoassay (EIA) with a rat antigalactomannan monoclonal antibody as capture and detector antibody. For each patient, sequential sera (3 to 20) were obtained before and after BMT. No positivity

A. Sulahian; M. Tabouret; P. Ribaud; J. Sarfati; E. Gluckman; J. P. Latgé; F. Derouin

1996-01-01

74

Detection of Galactomannan Antigenemia by Enzyme Immunoassay for the Diagnosis of Invasive Aspergillosis: Variables That Affect Performance  

Microsoft Academic Search

Invasive aspergillosis (IA) is a frequent complication of blood or marrow transplantation. Previous studies have reported that the Aspergillus galactomannan enzyme immunoassay (GM EIA) may be a useful diagnostic tool for IA, but its sensitivity is variable. We examined the performance of the GM EIA in 986 serum samples from 67 patients. Results demonstrated that decreasing the index cutoff for

Lisa McLaughlin; Marc Tabouret; Christopher Bentsen

2004-01-01

75

Determination of cortisol in saliva and serum by a luminescence-enhanced enzyme immunoassay.  

PubMed

A new luminescence-enhanced enzyme immunoassay (LEIA) has been developed and validated for the direct measurement of cortisol in saliva and serum. It has been demonstrated that this LEIA has a very good analytical and functional sensitivity. There was a good correlation to a commercial RIA. The assessment of diurnal cortisol profiles of healthy persons are discussed. Cortisol monitoring is indicated in diseases with abnormal glucocorticoid production such as Cushing's syndrome and Addison's disease. Because of the diurnal fluctuation of cortisol levels it is necessary to take several samples for an individual cortisol profile or during dynamic tests like the dexamethasone-suppression or ACTH stimulation. Salivary sample collection is an alternative method without the stress of repeated venipuncture. The measurement of cortisol in saliva is advisable in patients with abnormal cortisol-binding-globulin (CBG) levels such as pregnancy, hypothyroidism, nephrotic syndrome or marked adipositas and during the administration of certain drugs, especially oral contraceptives. PMID:15000217

Westermann, Jürgen; Demir, Anke; Herbst, Victor

2004-01-01

76

The European Sero-Epidemiology Network: standardizing the enzyme immunoassay results for measles, mumps and rubella.  

PubMed Central

The ESEN (European Sero-Epidemiology Network) project was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps and rubella in eight European countries. This involved achieving comparability both in the assay results from testing in different centres and also sampling methodology. Standardization of enzyme immunoassay results was achieved through the development of common panels of sera by designated reference centres. The panels were tested at the reference laboratory and then distributed to each participating laboratory for testing using their routine methods. Standardization equations were calculated by regressing the quantitative results against those of the reference laboratory. Our study found large differences in unitage between participants, despite all using an EIA method standardized against an international or local standard. Moreover, our methodology adjusted for this difference. These standardization equations will be used to convert the results of main serosurvey testing into the reference country unitage to ensure inter-country comparability. PMID:11057968

Andrews, N.; Pebody, R. G.; Berbers, G.; Blondeau, C.; Crovari, P.; Davidkin, I.; Farrington, P.; Fievet-Groyne, F.; Gabutti, G.; Gerike, E.; Giordano, C.; Hesketh, L.; Marzec, T.; Morgan-Capner, P.; Osborne, K.; Pleisner, A. M.; Raux, M.; Tischer, A.; Ruden, U.; Valle, M.; Miller, E.

2000-01-01

77

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

USGS Publications Warehouse

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

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

2001-01-01

78

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

PubMed

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

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

2011-01-01

79

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

80

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

PubMed Central

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

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

1991-01-01

81

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

PubMed

Amygdalin (laterile) is a cyanogenic glycoside commonly found in the pits of many fruits and raw nuts. When amygdalin-containing seeds are crushed and moistened, free cyanide is formed. Pits and nuts containing unusually high levels of amygdalin can therefore cause cyanide poisoning, and detection of amygdalin in food extracts can be a life-saving measure. In this study, we generated recombinant antibodies against amygdalin from a phage display of a combinatorial rabbit/human chimeric antibody library and used it in a sensitive competition enzyme immunoassay system to detect amygdalin in extracts of pits and nuts. The detection limit was determined to be 1 x 10(-9) M. PMID:18939751

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

2008-10-01

82

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

PubMed

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

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

2014-06-01

83

An enzyme immunoassay for the detection of Florida red tide brevetoxins.  

PubMed

A non-competitive solid-phase enzyme immunoassay for detection of brevetoxins in various matrices has been developed. The assay utilizes antibodies raised in a goat against brevetoxin PbTx-3-keyhole limpet hemocyanin conjugates with specific purification of brevetoxin antibodies through protein G and brevetoxin affinity columns, and rabbit anti-goat antibodies covalently linked to horseradish peroxidase. The assay was used specifically to detect brevetoxins in both cell culture and contaminated tissues. Sensitivity of the assay is 0.04 picomolar, and toxin can be quantified from 0.04 pM to 0.4 pM brevetoxin per well in microtiter plates by comparison with standard curves. PMID:1814015

Trainer, V L; Baden, D G

1991-01-01

84

Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural products.  

PubMed

In this work, a micro-plate chemiluminescence enzyme immunoassay by antibody-coated for the determination of aflatoxin B1 (AFB1) in agricultural products has been established. Aflatoxin B1 antibody (AFB1-Ab) was adsorbed physically on polystyrene micro-plate hole as solid phase antibody, which took place immunity-reaction between antigen and antibody with AFB1 standard solution or samples by direct competition. Luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase (HRP) with p-iodophenol enhancement was used as signal detecting system. The effects of several factors, including composition and pH of coating solution, dilution ratio and amount of antibody and enzyme labeled antigen, time of antibody-coating, incubation and chemiluminescence reaction, and other relevant variables upon the immunoassay were studied and optimized. The linear range of proposed method for AFB1 was 0.05-10.0 ng g(-1) with a correlative coefficient of -0.9997. The sensitivity of the proposed method was 0.01 ng g(-1). The RSDs of intra- and inter-assay were less than 12.2% and 10.0%, respectively. This method has been successfully applied to the evaluation of AFB1 in agricultural products with recoveries of 79.8%, 101.9% and 115.4% for low, middle and high concentration samples, respectively. It shows a good correlation with the commercial available ELISA kit for AFB1 with correlative coefficient of 0.9098 indicating that the established CLEIA method can be used to determine AFB1 in real samples. PMID:21315923

Fang, Luqiu; Chen, Hui; Ying, Xitang; Lin, Jin-Ming

2011-03-15

85

[[Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus].  

PubMed

To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were <10%, and the CoV of different ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV. PMID:25868272

Li, Jian-dong; Zhang, Quan-fu; Zhang, Shuo; Li, Chuan; Liu, Qin-zhi; Liang, Mi-fang; Li, De-xin

2014-11-01

86

Comparison of enzyme linked immunosorbent assay and enzyme linked fluorescence immunoassay for detection of antibodies against Chlamydia trachomatis.  

PubMed Central

An enzyme linked fluorescence immunoassay (ELFA) has been evaluated for the detection of antibodies against Chlamydia trachomatis. Reticulate bodies and elementary bodies from C trachomatis L2/434 strain were used as antigens. An enzyme linked immunosorbent assay (ELISA) has also been evaluated using the same antigens. Results obtained by ELISA and ELFA for human sera with these two antigens were compared with each other and with the results obtained by a micro-immunofluorescence (micro-IF) test. Serum IgG antibodies against C trachomatis L2 reticulate bodies and elementary bodies were found in 32 (20.0%) and 11 (6.9%), respectively, of 160 serum samples from pregnant women by the micro-IF test (titre greater than or equal to 1/32). All of these 32 pregnant women had IgG antibodies to C trachomatis reticulate bodies (titre greater than or equal to 1/100), whereas 20 (12.5%) had IgG antibodies to elementary bodies in the ELISA. On the other hand, 25 (15.6%) and 19 (11.9%) of them had IgG antibodies to C trachomatis L2 reticulate bodies and elementary bodies, respectively, by the ELFA (titre greater than or equal to 1/500). PMID:3882764

Numazaki, K; Chiba, S; Moroboshi, T; Kudoh, T; Yamanaka, T; Nakao, T

1985-01-01

87

Comparison of enzyme linked immunosorbent assay and enzyme linked fluorescence immunoassay for detection of antibodies against Chlamydia trachomatis.  

PubMed

An enzyme linked fluorescence immunoassay (ELFA) has been evaluated for the detection of antibodies against Chlamydia trachomatis. Reticulate bodies and elementary bodies from C trachomatis L2/434 strain were used as antigens. An enzyme linked immunosorbent assay (ELISA) has also been evaluated using the same antigens. Results obtained by ELISA and ELFA for human sera with these two antigens were compared with each other and with the results obtained by a micro-immunofluorescence (micro-IF) test. Serum IgG antibodies against C trachomatis L2 reticulate bodies and elementary bodies were found in 32 (20.0%) and 11 (6.9%), respectively, of 160 serum samples from pregnant women by the micro-IF test (titre greater than or equal to 1/32). All of these 32 pregnant women had IgG antibodies to C trachomatis reticulate bodies (titre greater than or equal to 1/100), whereas 20 (12.5%) had IgG antibodies to elementary bodies in the ELISA. On the other hand, 25 (15.6%) and 19 (11.9%) of them had IgG antibodies to C trachomatis L2 reticulate bodies and elementary bodies, respectively, by the ELFA (titre greater than or equal to 1/500). PMID:3882764

Numazaki, K; Chiba, S; Moroboshi, T; Kudoh, T; Yamanaka, T; Nakao, T

1985-03-01

88

Immunoassays of fungal laccases for screening of natural enzymes and control of recombinant enzyme production.  

PubMed

Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli. Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross-reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase-producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed. PMID:24112404

Loginov, Dmitry S; Vavilova, Ekaterina A; Savinova, ?lga S; Abyanova, Alfia R; Chulkin, Andrey M; Vasina, Daria V; Zherdev, Anatoly V; Koroleva, Olga V

2014-01-01

89

Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay.  

PubMed

Rapid-response analytical tests that can be performed at the point of sampling are based on a visual detection system. The influence of different factors on the signal generation in a membrane-based enzyme immunoassay was investigated. The research was applied to a flow-through immunoassay for the detection of ochratoxin A (OTA). This assay format is a very convenient, simple and fast qualitative screening tool. Conjugates of OTA with horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used as enzyme tracers. A new conjugate OTA-AP has been synthesized in our laboratory and its performance in the assay was compared with that of OTA-HRP. Different substrate systems for HRP and AP were compared. Several reagents, including polymers and surfactants, were tested for their possible effect on signal generation with the use of OTA-HRP conjugate. Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted a favourable effect on signal amplification, whereas surfactants negatively affected assay performance. The highest signal amplification (30-70% compared to the standard assay procedure) was achieved using 0.5% PVA in tetramethylbenzidine (TMB) Colorburst substrate solution and phosphate-buffered saline (PBS) for the washing step. It allowed more reliable visual estimation of the results from OTA-HRP assay. Exclusion of the detergent (Tween 20) from the washing solution exerted a favourable effect on assay performance using both enzyme tracers. The assay using OTA-HRP was more susceptible to matrix interferences than the assay with OTA-AP. Signal development in the matrix was better for the OTA-AP assay and visual estimation of the results was easier to perform in this case. For the analysis of spiked wheat samples, OTA-AP conjugate gave a more sensitive, stable and reproducible assay with a cut-off level of 4 microg kg(-1) for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples. PMID:17146620

Kolosova, Anna Yu; De Saeger, Sarah; Eremin, Sergei A; Van Peteghem, Carlos

2007-02-01

90

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

SciTech Connect

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

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

1987-06-29

91

Potential impact of different cytomegalovirus (CMV) IgM assays on an algorithm requiring IgM reactivity as a criterion for measuring CMV IgG avidity.  

PubMed

The measurement of cytomegalovirus (CMV) IgG avidity is a powerful tool for identifying individuals with recent CMV infection. Because such patients are expected to be positive for CMV IgM, several investigators have suggested that CMV IgG-positive sera first be screened for CMV IgM and then only the IgM-reactive sera be tested for avidity. We investigated the impact of different CMV IgM assays on such a reflexing algorithm using a panel of 369 consecutive IgG-positive serum samples submitted for avidity testing. A bead-based immunofluorescent assay (BIFA) identified 105 IgM-positive serum samples, whereas an IgM-capture enzyme immunoassay (EIA) identified 48 IgM-positive serum samples; this marked difference led us to evaluate additional CMV IgM assays. An enzyme-linked immunofluorescent assay (ELFA) and a chemiluminescent immunoassay (CIA) were used to test all sera with discordant BIFA/EIA results, all sera with concordant positive results, and selected sera with concordant negative results. The findings indicated that the ELFA would identify 74 CMV IgM-positive samples and the CIA would identify 64. Of the 23 low-avidity serum samples, 2 were IgM negative by BIFA, 3 by ELFA and CIA, and 4 by EIA; of the 23 intermediate-avidity serum samples, 6 were IgM negative by BIFA, 10 by ELFA, and 15 by EIA and CIA. In both these avidity groups, BIFA IgM-negative sera were also negative by the other 3 assays. These findings demonstrate that an algorithm requiring CMV IgM reactivity as a criterion for CMV IgG avidity testing does not identify all low-avidity sera and thus misses some cases of acute CMV infection. PMID:24671558

Prince, Harry E; Lapé-Nixon, Mary; Brenner, Andrew; Pitstick, Nancy; Couturier, Marc Roger

2014-06-01

92

Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids.  

PubMed Central

The monoclonal antibody solution hybridization assay is a novel enzyme immunoassay for detection of RNA with a biotinylated DNA probe. To increase the sensitivity of this test, a fluorescent substrate and an enzymatic amplification cycling system were compared with a conventional colorigenic substrate for alkaline phosphatase. The fluorescent, cycling, and colorigenic substrates detected, respectively, 10, 10, and 100 amol of unbound alkaline phosphatase in 2 h. With a prolonged incubation period of 16.6 h, the conventional substrate measured 10 amol of the enzyme. In the immunoassay for RNA detection, the fluorescence and cycling assays were faster than that using the colorigenic substrate and reached an endpoint sensitivity of 3.2 pg/ml (0.16 pg per assay) of cRNA. However, longer incubation periods (16.6 h) for optimal generation of the colorigenic product led to a comparable level of sensitivity for the conventional substrate. PMID:2473088

Coutlee, F; Viscidi, R P; Yolken, R H

1989-01-01

93

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

Microsoft Academic Search

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.

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

2005-01-01

94

Rapid detection of influenza A neuraminidase subtypes by cDNA amplification coupled to a simple DNA enzyme immunoassay  

Microsoft Academic Search

Summary A newly developed colorimetric method, DNA enzyme immunoassay (DEIA), was applied to the detection of neuraminidase subtypes N1 and N2 of influenza A viruses. Reverse transcription and polymerase chain reaction with universal primers were used for genomic amplification of H1N1, H2N2, and H3N2 strains. Following amplification, an aliquot of the PCR product was hybridized to biotinylated DNA sequences (N1\\/N2

B. Schweiger; I. Lange; R. Heckler; H. Willers; E. Schreier

1994-01-01

95

New Multiple Antigenic Peptide-Based Enzyme Immunoassay for Detection of Simian Immunodeficiency Virus Infection in Nonhuman Primates and Humans  

Microsoft Academic Search

Infections with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) are zoonotic infections. In Africa, the potential exists for additional cross-species transmissions from at least 33 different species of simian immunodeficiency virus (SIV)-infected nonhuman primates (NHPs) through hunting and butchering of these animals for food. Here we describe a highly sensitive and specific enzyme immunoassay (EIA) with

C. B. Ndongmo; W. M. Switzer; C. P. Pau; C. Zeh; A. Schaefer; D. Pieniazek; T. M. Folks; M. L. Kalish

2004-01-01

96

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

Microsoft Academic Search

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus,

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

2004-01-01

97

New neonatal thyrotropin enzyme immunoassay with fluorimetric detection: comparison with time-resolved fluoroimmunoassay.  

PubMed

This short communication compares a novel fluorimetric microplate enzyme immunoassay (FEIA) with a commercial time-resolved fluoroimmunoassay for the determination of thyrotropin in dried blood spots. The evaluation was performed using a retrospective study design with newborn blood samples from three screening centres. Non-parametric Spearman rank correlation analysis revealed highly significant positive correlation between methods: rs = 0.465, p < 0.0001 (Hannover), rs = 0.659, p < 0.0001 (Minsk), rs = 0.755, p < 0.0001 (Helsinki). Wilcoxon signed rank test performed for paired FEIA and time-resolved fluoroimmunoassay showed that the results obtained by both tests represented the same distribution (p < 0.0001). The new method, using fluorimetric detection, can be performed with the instrumentation commonly used for the screening of congenital hypothyroidism and phenylketonuria. Results are obtained within three to four hours after arrival of the sample in the laboratory. Preliminary evaluation indicates the method to be a suitable alternative to time-resolved fluoroimmunoassay for neonatal thyroid function screening. PMID:8439597

Tuuminen, T; Käpyaho, K I; Rakkolainen, A E; Bugrova, V B; Tsukerman, G L; Jesse, E; Sander, J

1993-01-01

98

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

PubMed

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

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

2014-08-15

99

Early diagnosis of typhoid fever by an enzyme immunoassay using Salmonella typhi outer membrane protein preparations.  

PubMed

An enzyme immunoassay (EIA) for detection of serum antibodies in patients with typhoid fever was developed using Salmonella typhi outer membrane protein (OMP) preparations as antigen. Acute phase (first week) sera from adult typhoid fever patients were tested as well as sera from the following control groups: adult travellers with diarrhea caused by enterotoxigenic Escherichia coli, children infected with Campylobacter jejuni, healthy Mexican adult blood donors, and adults with septicemia caused by other organisms. At a 1:3,125 serum dilution, the mean absorbance values were 1.41 in the typhoid fever patients, and 0.57, 0.55, 0.51 and 0.52 in the respective control groups. Inhibition EIA studies using OMP preparations or lipopolysaccharide (LPS) as free antigen indicated that proteins can play an important role in the detection of antibodies in early typhoid fever. This EIA may be useful for the diagnosis of typhoid fever since results were obtained within about five hours and in an endemic area antibodies against Salmonella typhi OMP preparations appear early in the course of the disease. PMID:8513812

Verdugo-Rodríguez, A; López-Vidal, Y; Puente, J L; Ruíz-Placios, G M; Calva, E

1993-04-01

100

[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

2012-01-01

101

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

PubMed

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

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

2014-04-15

102

Determination of methyl 2-benzimidazolecarbamate in bulk fruit juice concentrates by competitive-inhibition enzyme immunoassay.  

PubMed

A polyclonal enzyme immunoassay (EIA) was used to quantitate methyl 2-benzimidazolecarbamate (MBC or carbendazim), a degradation product of benomyl, in bulk fruit juice concentrates. These concentrates are used by industrial producers to prepare juice or juice concentrates sold in supermarkets. Total sample analysis time was less than 18 min without cleanup or 35 min with cleanup. As many as 8 samples can be analyzed simultaneously, with a limit of quantitation of 10 ppb. The assay's dynamic range ran from 0.5 to 20 ppb MBC but was best from 0.5 to 10 ppb. Intra-assay coefficients of variation (CVs) varied from 4.0 to 13% for standards and from 4.1 to 26% for samples. Interassay CVs varied from 4.5 to 47% for standards and 5.6 to 22% for samples. Average recovery of several juice concentrates spiked at 10 to 290 ppb was 97%. A total of 140 juice concentrates comprising 20 different kinds of juice were analyzed by 2 EIA methods and one liquid chromatographic (LC) procedure. MBC-positive samples gave the following correlation coefficients: 0.954 for EIA without cleanup vs LC, 0.956 for EIA with cleanup vs LC, and 0.978 for EIA with cleanup vs EIA without cleanup. MBC concentrations in MBC-positive juice samples ranged from 5 to 2960 ppb. PMID:7950422

Bushway, R J; Young, B E; Paradis, L R; Perkins, L B; Martin, S K; Brown, M P

1994-01-01

103

Detection of galactomannan antigenemia by enzyme immunoassay in experimental invasive aspergillosis.  

PubMed

A sensitive enzyme immunoassay (EIA) for galactomannan antigenemia that avoids the use of radioisotopes was devised. Three carbohydrate-rich antigenic fractions were purified from Aspergillus fumigatus 2085: a cold alkali extract (CA) from mycelium, an acetone-precipitated pyridine extract (APSK-66) from mycelium, and a methanol precipitate from culture filtrate. CA and APSK-66 were further purified by gel filtration and ion-exchange chromatography, respectively. An acid hydrolysate of CA contained only mannose and galactose, as determined by gas-liquid chromatography. Rabbit antisera were raised against conidia, mycelia, and cell walls of A. fumigatus. By indirect EIA, the best immunoglobulin G response (1/8,000) was obtained against CA in rabbits immunized intravenously with cell walls. Antigenemia was detected by indirect EIA inhibition in heat-dissociated sera of four immunosuppressed rabbits that were infected intravenously but was absent in two uninfected controls. The circulating antigen was resistant to pronase, was adsorbed onto concanavalin A, and had a molecular size of 50 to 100 kilodaltons. PMID:3294887

de Repentigny, L; Boushira, M; Ste-Marie, L; Bosisio, G

1987-05-01

104

Detection of galactomannan antigenemia by enzyme immunoassay in experimental invasive aspergillosis.  

PubMed Central

A sensitive enzyme immunoassay (EIA) for galactomannan antigenemia that avoids the use of radioisotopes was devised. Three carbohydrate-rich antigenic fractions were purified from Aspergillus fumigatus 2085: a cold alkali extract (CA) from mycelium, an acetone-precipitated pyridine extract (APSK-66) from mycelium, and a methanol precipitate from culture filtrate. CA and APSK-66 were further purified by gel filtration and ion-exchange chromatography, respectively. An acid hydrolysate of CA contained only mannose and galactose, as determined by gas-liquid chromatography. Rabbit antisera were raised against conidia, mycelia, and cell walls of A. fumigatus. By indirect EIA, the best immunoglobulin G response (1/8,000) was obtained against CA in rabbits immunized intravenously with cell walls. Antigenemia was detected by indirect EIA inhibition in heat-dissociated sera of four immunosuppressed rabbits that were infected intravenously but was absent in two uninfected controls. The circulating antigen was resistant to pronase, was adsorbed onto concanavalin A, and had a molecular size of 50 to 100 kilodaltons. PMID:3294887

de Repentigny, L; Boushira, M; Ste-Marie, L; Bosisio, G

1987-01-01

105

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

106

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

SciTech Connect

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

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

1986-07-18

107

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

108

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

PubMed

An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01pg/mL to 1.0ng/mL with a low detection limit of 6.0fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. PMID:25437365

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

2015-04-15

109

Comparison of Second- and Third-Generation Enzyme Immunoassays for Detecting Antibodies to Hepatitis C Virus  

PubMed Central

Supplemental assays, such as recombinant immunoblot assays (RIBA), are used to confirm detection of antibodies to hepatitis C virus (HCV). However, due to their expense, they are not widely used in developing countries. The purpose of our study was to compare the results of second- and third-generation (G2 and G3, respectively) enzyme immunoassays (EIAs) and to resolve discordant results by using a supplemental assay to assess the reliability of G2 and G3 EIAs to confirm anti-HCV antibody-positive results. We performed both G2 and G3 EIAs for anti-HCV antibodies on 1,134 serum samples collected during the 2nd year of a longitudinal community-based study in Egypt; 35 samples with discordant results were tested by Abbott Laboratories Micro-Particle Immunoassay (M-EIA) and RIBA. Viremia was determined with an in-house nested reverse transcriptase PCR (RT-PCR) to detect HCV RNA. Concordance between the two assays (G2/G3) was 96.9%; 87 (7.7%) samples were positive and 1,012 (89.2%) were negative by both assays. For 17 samples, the discordant results were G2 assay negative and G3 assay positive, and for 18 samples, the discordant results were G2 assay positive and G3 assay negative. Among the 17 G2 assay-negative and G3 assay-positive samples, 15 were M-EIA positive and 7 were PCR positive. Among the 18 G2 assay-positive and G3 assay-negative samples, 2 were M-EIA positive and none were PCR positive. RIBA results from 24 discordant samples showed 87.5% agreement with the G3 EIA, 12.5% agreement with the G2 EIA, and 95.8% agreement with M-EIA. Eleven samples were indeterminate by RIBA and excluded from this analysis. Based on RIBA results, the sensitivity of the G3 EIA was 99%, compared to 89.8% for the G2 EIA, while the specificity of the G3 EIA was 99.8%, compared to 98.9% for the G2 EIA. These results show that the reliability of the G3 EIA in screening these sera is excellent, and the G3 assay can be used in the absence of supplemental tests where resources are limited. RIBA appears not to have advantages over the less expensive M-EIA screening assay. The main disadvantage of RIBA is the occurrence of indeterminate results, especially among problematic samples. Samples giving discordant results in multiple assays are often indeterminate with the RIBA. PMID:11980937

Abdel-Hamid, Mohamed; El-Daly, Mai; El-Kafrawy, Sherif; Mikhail, Nabiel; Strickland, G. Thomas; Fix, Alan D.

2002-01-01

110

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 0.1-30 ng mL(-1) with the detection limit of 0.1 ng mL(-1). The linear detection range could match the concentration range within the "diagnostic gray zone" of serum f-PSA levels (4-10 ng mL(-1)). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL(-1)). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum. PMID:24139579

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

2013-11-01

111

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

PubMed

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

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

1991-02-01

112

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

PubMed Central

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

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

1994-01-01

113

Enzyme immunoassay for swine trichinellosis using antigens purified by immunoaffinity chromatography  

SciTech Connect

Various preparations of crude and a purified preparation of Trichinella spiralis antigens were compared in a rapid, micro-enzyme immunoassay (EIA) for detecting trichinellosis in swine. The crude antigen preparations (XM-300 or S/sub 3/ fraction) were lipid-free, cell-free fractions of muscle larvae, and the purified antigen was prepared by immunoaffinity chromatography of the soluble fraction of stichocyte secretory granules from rat muscle larvae. The antigens were tested against normal and immune swine sera for sensitivity and specificity, and for their ability to detect seroconversions early in the immune response. Tests of sequential sera from experimentally-infected pigs showed that the column antigen produced lower absorbances with pre-infection sera and, from 18 days post-infection, higher absorbances with positive sera. From 21-28 days post-infection, absorbances and S/N ratios with column antigen were nearly twice those with XM-300. Column antigen detected antibodies more often than XM-300 antigen in sera collected prior to the appearance of larvae. Crude antigen did not distinguish all true negatives from weakly positives in a study involving 100 sera from muscle digestion-negative pigs and 75 sera from experimentally infected pigs, whereas the column antigen distinguished all negatives from positives. In a larger scale test of the column antigen, 1130 pigs from Puerto Rico were tested in the micro-EIA test. Puerto Rico has no endogenous trichinellosis, and all 1130 pigs were shown to be muscle digestion negative. These results show that the column antigen out-performs the crude antigens in sensitivity, specificity, and early detection. The column antigen is therefore a major improvement in the EIA for swine trichinellosis.

Seawright, G.L.; Despommier, D.; Zimmermann, W.; Isenstein, R.S.

1983-11-01

114

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

PubMed

A power-free, portable "Chip EIA" was designed to render the popular Enzyme Linked Immunosorbent Assay (ELISA) more suitable for point-of-care testing. A number of microfluidic platforms have enabled miniaturization of the conventional microtitre plate ELISA, however, they require external pumping systems, valves, and electric power supply. The Chip EIA platform has eliminated the need for pumps and valves through utilizing a simple permanent magnet and magnetic nanoparticles. The magnetic nanoparticles act as solid support to capture the target and are then moved through chambers harboring different reagents necessary to perform a sandwich ELISA. The use of magnetic nanoparticles increases the volume-to-surface ratio reducing the assay time to 30 min. Changing the color of horseradish peroxidase (HRP) substrate to green indicates a positive result. In addition, a quantitative read-out was obtained through the use of cellphone camera imaging and analyzing the images using Matlab®. Cell phones, including smart ones, are readily available almost everywhere. The Chip EIA device was used to assay total prostate specific antigen (tPSA) in 19 serum samples. The PSA Chip EIA was tested for accuracy, precision, repeatability, and the results were correlated to the commercial Beckman Colter, Hybritech immunoassay® for determination of tPSA in serum samples with a Pearson correlation coefficient (R(2)=0.96). The lower detection limit of the PSA Chip EIA was 3.2 ng/mL. The assay has 88.9% recovery and good reproducibility (% CV of 6.5). We conclude that the developed Chip EIA can be used for detection of protein biomarkers in biological specimens. PMID:23811482

Adel Ahmed, Heba; Azzazy, Hassan M E

2013-11-15

115

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody.  

PubMed

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2680124

Hosoda, H; Tsukamoto, R; Nambara, T

1989-07-01

116

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

PubMed Central

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

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

2003-01-01

117

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

118

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

PubMed Central

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

DeLuca, Jane

2007-01-01

119

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

120

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

PubMed Central

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

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

2014-01-01

121

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

PubMed

A single-step, easy-to-use enzyme immunoassay capillary sensor, composed of functional multilayer coatings, was developed in this study. The coatings were composed of substrate-immobilized hydrophobic coating, hydrogel coating, and soluble coating containing an enzyme-labeled antibody. The response mechanism involved a spontaneous immunoreaction triggered by capillary action-mediated introduction of a sample antigen solution and subsequent separation of unreacted enzyme-labeled antibodies and antigen-enzyme-labeled antibody complexes by the molecular sieving effect of the hydrogel. An enzyme reaction at the substrate-immobilized hydrophobic coating/hydrogel coating interface resulted in a protein-selective fluorescence response. An antigen concentration-dependent response was obtained for diagnostic marker protein samples (hemoglobin A1c (HbA1c), 7.14-16.7 mg mL(-1); alpha-fetoprotein (AFP), 1.4-140 ng mL(-1); C-reactive protein (CRP), 0.5-10 ?g mL(-1)) that cover a clinically important concentration range. The successful measurement of CRP in diluted serum samples demonstrated the application of this capillary sensor. PMID:25599100

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

2015-03-01

122

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

PubMed Central

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

Takehara, Shizuka; Takahashi, Masaharu

2013-01-01

123

An enzyme-linked chemiluminescent immunoassay developed for detection of Butocarboxim from agricultural products based on monoclonal antibody.  

PubMed

In this study, four different haptens around the oxime moiety of Butocarboxim were designed and synthesised. Two of the haptens were conjugated with bovine serum albumin (BSA) to serve as the immunogen and all the haptens were conjugated with ovalbumin (OVA) for the coating antigen. The anti-Butocarboxim monoclonal antibody (Mab) was selected based on eight immunogen/coating antigen combinations. The first enzyme-linked chemiluminescent immunoassay (ELCIA) for determining Butocarboxim in agricultural products was developed. Under the optimised conditions, the detection limit for the ELCIA was 20 ng·mL(-1) and the linear range was 27-2700 ng·mL(-1). Analyte recoveries for extracts of spiked agricultural (apple and greengrocery) products and tap water ranged from 97.18% to 107.00%. The developed immunoassay has great potential to be developed as a test kit offering a simple and cost-effective approach (such as lateral flow test strip) for screening purposes and evaluating environmental exposure to Butocarboxim. PMID:25053070

Fang, Qingkui; Wang, Limin; Hua, Xiude; Wang, Yulong; Wang, Suyan; Cheng, Qi; Cai, Jia; Liu, Fengquan

2015-01-01

124

A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays.  

PubMed

Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making. PMID:11305642

Grigorenko, V; Andreeva, I; Börchers, T; Spener, F; Egorov, A

2001-03-15

125

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

126

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

Microsoft Academic Search

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

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

2002-01-01

127

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

Microsoft Academic Search

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

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

128

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

129

A new enzyme-linked fluorescence assay (ELFA) for use with peroxidase-antibody conjugates: a comparison with ELISA for the quantitation of IgM antibodies to hepatitis B core antigen.  

PubMed

A new enzyme-linked fluorescence assay (ELFA) suitable for use with peroxidase-antibody conjugates is described. The substrate for the assay is p-hydroxyphenylacetic acid, the fluorescent product of which is stable and unaffected by light. The assay compared favourably with a standard ELISA for the quantitation of IgM antibodies to hepatitis B core antigen. PMID:3517341

Dave, J R; Taylor, P; Grange, J M; Gaya, H

1986-05-01

130

[Investigation of a new HIV-1 p24 antigen detection kit based on the enzyme-linked fluorescent immunoassay].  

PubMed

We investigated the performance of the new p24 antigen detection kit (VIDAS HIV p24) with the conventional antigen kit (HIV-1 Ag monoclonal; Abbott). The new kit is an enzyme-linked fluorescent immunoassay (ELFA) and all of the assay steps are performed automatically by the VIDAS instrument within 100 minutes. With the seven HIV-1 seroconversion panels, three seroconversions were detected on an average of 6.8 days earlier with ELFA than the conventional EIA kit. ELFA showed negative results for all of the 11 false positive samples by the combined (p24, anti-HIV) detection kit (VIDAS HIV DUO). The results obtained suggest that ELFA are very useful for an earlier diagnosis of HIV infection and re-test for false positive samples by other HIV diagnosis kits. PMID:11068364

Hayashi, T; Saito, T; Kondo, M; Watanabe, S; Imai, M

2000-09-01

131

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

USGS Publications Warehouse

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

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

1987-01-01

132

Determination of phenytoin in plasma: a comparison of procedures using a new radioimmunoassay, gas chromatography, and enzyme immunoassay.  

PubMed

A new commercially available radioimmunoassay (RIA) procedure for the anticonvulsant drug phenytoin has been assessed and compared with a gas-liquid chromatographic (GLC) and an enzyme immunoassay (EMIT) procedure. One hundred and thirty-four plasma samples containing phenytoin were analyzed by the three methods and the results were found to be essentially independent of the procedure used and so would indicate similar clinical interpretation. The correlation coefficients were: RIA/GLC = 0.988; EMIT/RIA = 0.978; EMIT/GLC = 0.981. The cross reactivity of 33 drugs and related compounds was tested in the RIA and EMIT procedures and only a few very closely related substances showed significant cross reactivity. In addition, the three methods were compared with respect to the technical difficulties involved as well as time and cost for the assay of single and multiple samples. PMID:354921

Stanley, P E; Peikert, M R

1978-06-01

133

Determination of opiates and cocaine in hair using automated enzyme immunoassay screening methodologies followed by gas chromatographic–mass spectrometric (GC–MS) confirmation  

Microsoft Academic Search

The objective of this study was to develop a two-step strategy for analysis of opiates and cocaine in hair samples involving an immunological screening procedure followed by confirmation of results using gas chromatography–mass spectrometry (GC–MS). A semi-quantitative automated competitive enzyme-linked immunosorbent assay (ELISA) methodology using Oral Fluid Micro-Plate Enzyme Immunoassays (Orasure Technologies, Inc.) was developed and validated. Applicability was proven

Katrin Lachenmeier; Frank Musshoff; Burkhard Madea

2006-01-01

134

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

PubMed

On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells. PMID:25793322

Yang, Xinjian; Gao, Zhiqiang

2015-04-25

135

Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II.  

PubMed

This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species. PMID:15465665

Wilkinson, Ryan J; Elliott, Phillip; Hohmann, Art; Francis, Geoffrey; Carragher, John

2004-10-01

136

Evaluation of the Architect Epstein-Barr Virus (EBV) viral capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear antigen 1 IgG chemiluminescent immunoassays for detection of EBV antibodies and categorization of EBV infection status using immunofluorescence assays as the reference method.  

PubMed

Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The ? values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P < 0.0001) for VCA IgM, 0.889 (P < 0.0001) for VCA IgG, and 0.961 (P < 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states. PMID:24623623

Corrales, Isabel; Giménez, Estela; Navarro, David

2014-05-01

137

A subtype-specific peptide-based enzyme immunoassay for detection of antibodies to the G protein of human respiratory syncytial virus is more sensitive than routine serological tests.  

PubMed Central

Peptides deduced from the central conserved region (residues 158 to 189) of protein G of human respiratory syncytial virus (HRSV) subtypes A and B were used as antigens in subtype-specific enzyme-linked immunosorbent assays (G-peptide ELISAs). These G-peptide ELISAs were compared with seven other serological assays to detect HRSV infection: ELISAs based on complete protein G, on fusion protein F, and on nucleoprotein N; a complement fixation assay; a virus neutralization test; and ELISAs for the detection of immunoglobulin A (IgA) or IgM antibodies specific for HRSV. In paired serum samples from patients with HRSV infection, more infections were diagnosed by the G-peptide ELISA (67%) than by all other serological tests combined (48%). Furthermore, for 16 of 18 patients (89%), the G-peptide ELISAs were able to differentiate between antibodies against HRSV subtypes A and B. This study shows that peptides corresponding to the central conserved region of the attachment protein G of HRSV can successfully be used as antigens in immunoassays. The G-peptide ELISA appeared to be more sensitive than conventional tests for the detection of HRSV antibody titer rises. PMID:9196168

Langedijk, J P; Brandenburg, A H; Middel, W G; Osterhaus, A; Meloen, R H; van Oirschot, J T

1997-01-01

138

Serological Evaluation of Thin-Layer Immunoassay–Enzyme-Linked Immunosorbent Assay for Antibody Detection in Human Trichinellosis  

PubMed Central

A new immunoenzymatic test, named the thin-layer immunoassay–enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 ?l each of antigen (7 ?g/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis. PMID:10973459

Gómez-Priego, Alberto; Crecencio-Rosales, Lidia; de-la-Rosa, Jorge-Luis

2000-01-01

139

Evaluation of atrazine soil extraction methods for the determination by enzyme immunoassay and gas chromatography.  

PubMed

Four soil extraction methods were evaluated for the determination of atrazine and other s-triazines by ELISA and GC, using both field-treated and laboratory fortified samples. The most efficient recoveries for atrazine, simazine, and cyanazine from loam soil fortified at concentrations from 0.01 ppm to 1 ppm were obtained by mechanical wrist-action shaker (1 h) using methanol:water and solid phase extraction (SPE) cleanup (standard method). A handshaking extraction (1 min) with acetonitrile:water showed fairly good correlation with the standard extraction method and is suitable for field use with ELISA. Sonication using acetonitrile:water and SPE cleanup was the most efficient extraction method for the dealkylated metabolites (deisopropyl and deethyl atrazine) with recoveries higher than 60%. In general, supercritical fluid extraction (SFE) was as efficient as sonication and handshaking but was more variable. A guideline for validation of immunoassays and methods comparison is given. The sensitivity of the ELISA method was comparable to the GC and was both accurate and precise. Comparison of ELISA and GC determinations of 120 field soil samples and 40 laboratory spiked soil samples extracted with four different methods showed no false negatives or positives with excellent correlations and showed not significant differences (P > 0.05). An evaluation of the cost for GC and ELISA methods was also conducted. PMID:7944554

Del Valle, P L; Nelson, J O

1994-10-01

140

Detection of membrane-bound enzymes in cells using immunoassay and Raman microspectroscopy.  

PubMed

The method of surface-enhanced Raman microspectroscopy was developed for direct detection of membrane-bound enzymes in cells. Cells were cultured, fixed, and incubated with specific primary antibodies and their corresponding labeled secondary antibodies, and surface-enhanced Raman scattering (SERS) was detected directly in the wells of a multiwell plate. First, specific primary antibodies were separately bound to enzymes in cells. Then, the peroxidase-labeled secondary antibodies were added to bind these primary antibodies. Peroxidase substrates, o-phenylenediamine and hydrogen peroxide, were added and reacted for 15 min at room temperature to form azoaniline, a compound with strong Raman scattering. Then, Raman scattering of this enzymatic product was enhanced by silver colloids. Samples were excited with a He/Ne laser at 632.8 nm and SERS was detected by a CCD camera. The SERS spectrum of this product showed an intense peak at 1370 cm-1 and its intensity was used for assessment of cellular enzymes. The observed amount of enzyme was normalized to protein content in each well. The method was successfully used to detect prostaglandin H synthase-1 and -2 (PGHS-1 and -2) in normal human hepatocytes and human hepatocellular carcinoma (HepG2) cells. The detection limit of these PGHS enzymes by this method was about 0.1 pg per well. An immunohistochemical staining was also used to detect the expression of both PGHS isozymes in these cells. PMID:9618199

Hawi, S R; Rochanakij, S; Adar, F; Campbell, W B; Nithipatikom, K

1998-06-01

141

Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM.  

PubMed

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V-IgM and -IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V-specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non-infected, and 29 from other viral recent infections (Epstein-Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well-established Biotrin EIAs. PMID:23763266

de Ory, Fernando; Minguito, Teodora; Echevarría, Juan Emilio; Del Mar Mosquera, María; Fuertes, Antonio

2014-03-01

142

Multicenter Evaluation of New Double-Antigen Sandwich Enzyme Immunoassay for Measurement of Anti-Human Immunodeficiency Virus Type 1 and Type 2 Antibodies  

Microsoft Academic Search

A new enzyme immunoassay (EIA), the Cobas Core Anti-HIV-1\\/HIV-2 EIA DAGS (also referred to as Roche DAGS), for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 was evaluated in four centers. The assay is based on the double-antigen sandwich (DAGS) format, which enables the detection of all classes of antibodies. The antigens consist of recombinant

PHILIPPE BURGISSER; FRANCOIS SIMON; MARTIN WERNLI; THOMAS WUST; MARIE-FRANCOISE BEYA; ANDPHILIPPE C. FREI; Regionales Blutspendezentrum; Groupe Hospitalier; Bichat-Claude Bernard

1996-01-01

143

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

144

A Highly Sensitive Sandwich Enzyme Immunoassay for Insulin in Human Serum Developed Using Capybara Anti-Insulin Fab' Horseradish Peroxidase Conjugate  

Microsoft Academic Search

A highly sensitive sandwich enzyme immunoassay for insulin in human serum has been developed using capybara anti-insulin serum. Capybara anti-insulin IgG-coated polystyrene balls were incubated with serum samples in the presence of 0.4 M NaCl and then with capybara anti-insulin Fab'-horseradish peroxidase conjugate.The peroxidase activity bound to the polystyrene balls was correlated to the amount of insulin to be assayed.

Masayoshi Imagawa; Seiichi Hashida; Eiji Ishikawa; Hiroyuki Mori; Chiharu Nakai; Yutaka Ichioka; Katsuyuki Nakajima

1983-01-01

145

Two Enzyme Immunoassays and PCR for Detection of Helicobacter pylori in Stool Specimens from Pediatric Patients before and after Eradication Therapy  

Microsoft Academic Search

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect Helicobacter pylori antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine H. pylori-infected children with dyspepsia received eradication therapy. Successful treatment was determined by a

ATHANASIOS MAKRISTATHIS; WOLFGANG BAROUSCH; EVA PASCHING; CHRISTA BINDER; CHRISTA KUDERNA; PETRA APFALTER; MANFRED L. ROTTER; ALEXANDER M. HIRSCHL

2000-01-01

146

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

Microsoft Academic Search

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

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

147

Imaging of Lactobacillus brevis Single Cells and Microcolonies without a Microscope by an Ultrasensitive Chemiluminescent Enzyme Immunoassay with a Photon-Counting Television Camera  

Microsoft Academic Search

An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-mm pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol

TETSUJI YASUI; KUMIKO YODA

1997-01-01

148

Measurement of Intact Rat Osteocalcin in Osteoblast (ROS17\\/2.8) Cells and in Ovariectomized Rats with a Sandwich Enzyme Immunoassay  

Microsoft Academic Search

.   We developed a sandwich enzyme immunoassay system for intact rat osteocalcin to improve the region specificity for the detection\\u000a of this molecule. We synthesized two peptides of N-terminal 20 residues and C-terminal 10 residues of rat osteocalcin. After\\u000a conjugating these peptides with carrier protein, we obtained anti-N- and anti-C-terminal rat osteocalcin antibodies in rabbits\\u000a raised against these two peptides,

T. Ohta; Y. Azuma; M. Kiyoki; H. Eguchi; M. Arita; K. Hosoda; Y. Tsukamoto; T. Nakamura

1996-01-01

149

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

150

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

151

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

2007-09-01

152

Rapid and specific enzyme immunoassay on hydrophobic grid membrane filter for detection and enumeration of thermophilic Campylobacter spp. from milk and chicken rinses.  

PubMed

Six commercially available anti-Campylobacter antibodies were examined for their applicability in an enzyme immunoassay on hydrophobic grid membrane filters, both for the detection and enumeration of Campylobacter spp. When a panel of nine Campylobacter (seven Campylobacter jejuni and two Campylobacter coli) and eight non-Campylobacter strains were used in a dot-blot format enzyme immunoassay to test the specificity of these antibodies, only one polyclonal antibody (Biodesign) detected all Campylobacter strains. Escherichia coli O157:H7 produced weak nonspecific signals due to endogenous peroxidase activity. The specificity of this Biodesign antibody was further tested against 30 more Campylobacter strains and more than 600 non-Campylobacter strains on hydrophobic grid membrane filters grown on modified Campylobacter agar with charcoal and deoxycholate, a Campylobacter selective medium. All the Campylobacter strains were detected, whereas only two (Acinetobacter calcoaceticus, Salmonella Minnesota) of the approximately 130 non-Campylobacter strains, which grew on modified Campylobacter agar with charcoal and deoxycholate, gave false-positive signals. This simple, rapid, and specific enzyme immunoassay also detected Campylobacter spp. from inoculated milk and chicken rinses and naturally contaminated chicken rinses. PMID:10772214

Wang, H; Boyle, E; Farber, J

2000-04-01

153

Comparative analysis of tacrolimus (FK506) in whole blood liver transplant recipients by PRO-TRAC enzyme-linked immunosorbent assay and microparticle enzyme immunoassay IMX methods.  

PubMed

The macrolide tacrolimus (FK506) is a powerful immunosuppressive drug that acts early in the T-cell activation process and inhibits cytokine gene transcription. Data from several trials in liver transplantation have shown the efficacy of tacrolimus in the prevention of allograft rejection and its potent hepatotrophic effect, which could explain its great success in liver transplantation. However, tacrolimus is not devoid of adverse effects (mainly nephrotoxicity and neurotoxicity) requiring careful blood level monitoring, which is an essential aid in the adjustment of drug dosing. Several methods of analysis are available to measure tacrolimus in whole blood. A new assay based on the enzyme-linked immunosorbent assay (ELISA) technology has been developed. The INCSTAR PRO-TRAC FK506 is a sensitive immunoassay (range, 0.5 to 60 ng/ml), which uses a mouse monoclonal antibody to FK506. Samples are extracted into methanol and dried under nitrogen. The reconstituted extracts are analyzed by ELISA by using 2-h incubation. The aim of this study was to evaluate the ELISA method in routine monitoring of liver transplant patients and to compare the whole blood results with those obtained by Abbott microparticle enzyme immunoassay (MEIA) IMx. Precision studies with 20 samples from 4.37 and 17.1 ng/ml gave within-run total coefficients of variance of 14.4 and 17.4%, respectively. A total of 63 blood samples was analyzed. The mean +/- SD were 9.68 +/- 5.92 and 10.52 +/- 7.54 ng/ml by ELISA and MEIA assays, respectively. There was an acceptable correlation between the methods: ELISA = 1.419 + 0.785 MEIA; Sy x x = 2.639; r = 0.804. Serial tacrolimus measurements (n = 13) in two patients with bilirubin levels > 20 mg/dl yielded mean +/- SD (range) of 11.64 +/- 7.59 ng/ml (2.60-25.40 ng/ml) and 15.55 +/- 10.78 ng/ml (3.60-34.4 ng/mL) by ELISA and MEIA assays, respectively. These discrepancies in concentrations can result from variation in matrix or different cross-reactivities or both in the two tests. We concluded that the INCSTAR PRO-TRAC FK506 is suitable for routine whole blood tacrolimus monitoring. PMID:8946669

Brunet, M; Pou, L; Torra, M; López, R; Rodamilans, M; Corbella, J

1996-12-01

154

Development of miniaturized immunoassay: influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot.  

PubMed

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays. PMID:20079705

El Khoury, Graziella; Laurenceau, Emmanuelle; Chevolot, Yann; Mérieux, Yves; Desbos, Agnès; Fabien, Nicole; Rigal, Dominique; Souteyrand, Eliane; Cloarec, Jean-Pierre

2010-05-01

155

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

2011-09-01

156

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

PubMed

To protect public health from bovine spongiform encephalopathy, European Commission Regulation EC 1139/2003 on monitoring programs and specified risk material requires that as of 1 October 2003, each member state has in place a sampling plan with an appropriate laboratory test to detect central nervous system (CNS) tissue in bovine head meat harvested at slaughterhouses or cutting plants. With this study, we wanted to evaluate the accuracy and reliability of an enzyme immunoassay, the RIDASCREEN Risk Material 10/5, in targeting a CNS-specific marker, the glial fibrillary acidic protein. A receiver operating characteristics curve was plotted to identify the best cutoff of CNS concentration. Reliability was calculated by Cohen's kappa on data from two diagnostic sessions. Test performance showed high sensitivity and specificity (97.9 and 97.4%, respectively) for a cutoff value between positive and negative at a CNS concentration of 0.049%; reliability of test precision was also very good. When these criteria are applied, the RIDASCREEN Risk Material 10/5 test appears to be a reliable tool for monitoring CNS tissue contamination in meat. This diagnostic procedure should therefore be recommended for national application in monitoring programs. PMID:16995540

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

2006-09-01

157

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

PubMed Central

Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive microscopic reexamination. The remaining two specimens were from patients who previously tested positive and who had recurrent symptoms of or responded to therapy for giardia. Therefore, we consider both cases to be true positives. One specimen exhibited a single cyst by microscopic exam and was negative by EIA Resolved results yielded a relative sensitivity of 95%, a relative specificity of 100%, a positive predictive value of 99.6%, and a negative predictive value of 100%, compared with a sensitivity of 74% for conventional microscopy. PMID:7929778

Scheffler, E H; Van Etta, L L

1994-01-01

158

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

Microsoft Academic Search

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

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

2011-01-01

159

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

Microsoft Academic Search

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

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

2012-01-01

160

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins  

Microsoft Academic Search

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other\\u000a undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk\\u000a antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance\\u000a as one

H Pichler; S Baumgartner; M Freudenschuss; S Palme; EM Binder; R Krska

2001-01-01

161

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. PMID:2764563

Yao, R C; Mahoney, D F

1989-01-01

162

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

163

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

PubMed

The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an 'in-house' EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [(3)H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6 months of age and when between 6 and 24 months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas. PMID:22634955

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

2012-09-01

164

Comparison of enzyme- and radio-immunoassay kits to detect hepatitis B surface antibody response among hepatitis B vaccinees.  

PubMed

A total of 288 of sera was obtained from 96 hepatitis B vaccinees to test antibody to hepatitis B surface antigen (anti-HBs) by enzyme immunoassay (EIA) and radioimmunoassay (RIA), respectively. An anti-HBs titer greater than 10 mIU/mL is required for immunity; 172 (59.7%) were positive by EIA and 173 (60.1%) were positive by RIA. A correlation study of anti-HBs titers tested by these two methods showed a correlation coefficient r = 0.889 (n = 162, p less than 0.001). Of 173 serum specimens whose anti-HBs were positive by RIA, 10 were negative by EIA; those 10 had had anti-HBs titers between 11 to 20 mIU/mL by RIA. Among 172 serum specimens whose anti-HBs were positive by EIA, 9 were negative by RIA; 3 of those were between 21 to 70 mIU/mL, and 6 were between 11 to 20 mIU/mL by EIA. Of the 96 vaccinees, following first dose of hepatitis B vaccination their anti-HBs response rates were found to be 20.8% by EIA and 17.7% by RIA. Following second and third vaccinations, their anti-HBs response rates were 65.6% and 92.7% by EIA, and 71.9% and 90.6% by RIA, respectively. Thus, EIA correlates well with RIA for anti-HBs testing especially when its titers are greater than 70 mIU/ml. PMID:3072155

Lee, S D; Lo, K J; Tsai, Y T; Chan, C Y; Lin, H C; Wu, J C

1988-08-01

165

Comparison of Hemagglutination Inhibition Assay and Enzyme Immunoassay for Determination of Mumps and Rubella Immune Status in Health Care Personnel  

PubMed Central

Background Screening tests are available to determine immunity to vaccine-preventable diseases, such as mumps and rubella. We aimed to define better assay for detecting immune status of health care personnel to vaccine-preventable diseases. Methods Mumps and rubella antibodies of health care personnel at Shimane University Hospital were examined by hemagglutination inhibition assay (HI), comparing with those by enzyme immunoassay (EIA). Results A total of 910 sera from health care personnel were tested. There was poor correlation between HI and EIA in detecting mumps antibodies with correlation coefficient values (r) = 0.190 (P < 0.001), but in rubella antibodies HI and EIA were relatively well correlated (r = 0.930, P < 0.001). Seropositivity rate of HI versus EIA was found to be 65.7 versus 93.2, and 89.5 versus 86.5% for mumps and rubella, respectively. As compared with EIA, HI identified sixfold larger seronegative subjects in mumps. Moreover, in mumps, 88.8% of seronegative subjects detected by HI were seropositive by EIA, while 3.7% of seropositive subjects detected by HI were seronegative by EIA. In rubella, 2.1% of seronegative subjects detected by HI were seropositive by EIA, and 1.7% of seropositive by HI was seronegative by EIA. Conclusion Considerable difference between HI and EIA in determining immune status of health care personnel to mumps and rubella suggests beneficial use of EIA for the identification of accurate susceptible personnel who subsequently undergo an effective vaccination programs. Seroprevalence survey of health care personnel by using appropriate assay is essential for prevention and infection control strategies in health care settings. PMID:24038230

Kumakura, Shunichi; Shibata, Hiroshi; Isobe, Takeshi; Hirose, Masahiro; Ohe, Miki; Nishimura, Nobuhiro; Onoda, Keiichi; Nagai, Atsushi; Yamaguchi, Shuhei

2013-01-01

166

Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection of Mycobacterium tuberculosis Infection  

PubMed Central

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-?) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-? per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-?. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-?s. Cross-reactions with other human cytokines or with IFN-?s derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-? in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-? were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-? responsiveness in vitro. These results support the further study of the blood culture–IFN-? EIA system as an alternative to skin testing for the detection of human TB infection. PMID:9665962

Desem, Nuket; Jones, Stephen L.

1998-01-01

167

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

PubMed

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

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

2014-09-01

168

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

2013-03-30

169

The effect of storage conditions on salivary cortisol concentrations using an enzyme immunoassay.  

PubMed

Saliva samples are easy to collect and are applicable for home-sampling, e.g. when studying HPA-axis dynamics to characterize diurnal cortisol profiles and the cortisol awakening response. However, the storing and transport conditions might be critical in the home-sampling approach. Here, we tested the stability of saliva cortisol in samples stored at different temperatures and after repeated thawing-freezing cycles when measured with an Enzyme Immuno Assay (EIA). Thirteen healthy volunteers, six women and seven men, mean age 31 (range 26-49) years collected saliva either in the morning hours (08:00-10:00 h) or before lunch (11:00-12:00 h). Storage at six different conditions were tested: Storage at - 18°C, - 4°C, 4°C and room temperature for 72 h. One condition tested was at - 18°C for 72 h and then kept in an envelope for 72 h with a freezing element in room temperature surroundings where after it was stored at - 80°C. The last tube was stored directly at - 80°C and served as the 'gold standard'. The saliva samples were assayed using Salivary Cortisol Diagnostic EIA. Differences in cortisol measurements between each of the five conditions and the 'gold standard' (- 80°C) were evaluated by one-sample t-test. No significant differences were observed. This indicates that an EIA method can be used reliably when measuring salivary cortisol samples obtained by home-sampling including a postal delivery. PMID:25510953

Nalla, Anjana A; Thomsen, Gerda; Knudsen, Gitte M; Frokjaer, Vibe G

2015-01-01

170

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

SciTech Connect

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

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

1996-10-01

171

Development and application of an immunoaffinity column enzyme immunoassay for mycotoxin zearalenone in complicated samples.  

PubMed

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

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

2014-01-01

172

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

2013-09-27

173

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

PubMed Central

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

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

2014-01-01

174

Enzyme-linked immunoassay for human immunodeficiency virus type 1 envelope glycoprotein 120.  

PubMed Central

An enzyme-linked immunosorbent assay (ELISA) that can measure picogram quantities of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) in cell culture medium or body fluids has been developed. Recombinant, soluble CD4 immobilized in microtiter trays was used to capture gp120, which was then detected with polyclonal sheep antibody to gp120 followed by biotinylated rabbit anti-sheep immunoglobulin G and an avidin-alkaline phosphatase indicator system. With a reference recombinant gp120, the assay showed a linear relationship between optical density and concentrations ranging from 60 to 6,000 pg/100-microliters well; precision of the assay varied with the concentrations and ranged from +/- 40% with amounts smaller than 200 pg to +/- 10% with amounts larger than 200 pg. In a group of coded samples containing 60 pg (approximately 10(7) molecules) of reference gp120, the assay correctly identified the samples as containing gp120 99% of the time, with no false-positive results recorded for blank samples. Recombinant gp120 prepared in another cell culture system demonstrated a binding coefficient 13-fold lower than that of reference gp120. Mixing standard amounts of reference gp120 with increasing concentrations of human sera reduced assay sensitivity, although the linear relationship between gp120 concentration and optical density remained. With this assay we were able to detect gp120 in HIV-1 suspensions prepared from cultured lymphoblastoid cells and in the sera of HIV-1-infected patients. This ELISA for gp120 should be useful for studying the biological role of gp120 in HIV infection. PMID:1993748

Gilbert, M; Kirihara, J; Mills, J

1991-01-01

175

Microtiter virus isolation and enzyme immunoassays for detection of bovine viral diarrhea virus in cattle serum.  

PubMed Central

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in cattle herds. Two procedures for using serum to detect PI cattle were developed and evaluated. BVDV was found to remain viable for 7 days in serum samples stored at room temperature. The tests use cell culture virus isolation (VI) in 96-well microtiter plates, followed by immunostaining of cell monolayers with monoclonal antibodies. One technique, the immunoperoxidase monolayer assay (IPMA), forms a red intracellular precipitate while the other, the monolayer enzyme-linked immunosorbent assay (M-ELISA) produces a yellow color in solution. The optimal incubation period for microtiter VI was determined to be 4 days. Optimal IPMA staining was obtained by fixing cell monolayers with 20 to 30% acetone, whereas a simple dry-rehydrate-dry cycle provided optimal M-ELISA staining. The M-ELISA and IPMA had the same sensitivities and specificities, but the M-ELISA was a more rapid procedure and use of a spectrophotometer for reading samples allowed for greater objectivity. When compared to standard VI with routine samples submitted for the diagnosis of BVD, M-ELISA and IPMA had a relative sensitivity of 85% and a relatively specificity of 100%. When only samples from cattle suspected of being PI were considered, these two parameters were 100% for both IPMA and M-ELISA. The two procedures, especially the M-ELISA, are suitable for whole-herd testing to identify PI cattle. The appeal of these tests is derived from the convenience of using serum as a diagnostic sample and the ability to rapidly screen large numbers of samples at low cost. PMID:9157132

Saliki, J T; Fulton, R W; Hull, S R; Dubovi, E J

1997-01-01

176

A new enzyme immunoassay for aconitine and its application to quantitative determination of aconitine levels in plasma.  

PubMed

A reliable enzyme immunoassay (EIA) method was developed for quantitative determination of aconitine with high sensitivity and specificity. The bovine serum albumin (BSA)- and beta-galactosidase (beta-Gal) conjugates as immunogens and enzyme-labeled antigens were prepared by coupling of their proteins with succinic acid (short chain length; n=2, where n represents the number of methylene units) and hexadecanedioic acid (long chain length; n=14) hemiesters of benzoylaconine through the respective N-hydroxysuccinimide esters as intermediates. Two types of the BSA-conjugates with short and long chains were repeatedly injected into rabbits to obtain anti-aconitine antisera (As1 and As2, respectively). All combinations of beta-Gal-labeled antigens LAg1 (n=2) and LAg2 (n=14) with antisera As1 (n=2) and As2 (n=14) showed high sensitivity to aconitine in a range of 0.1-1.0 ng. Although the combination of LAg2 (n=14) with antiserum As1 (n=2) showed high specificity to aconitine, the combination of LAg2 (n=14) and As2 (n=14) was highly specific to both aconitine and mesaconitine. When aconitine was intravenously administered to rats, the aconitine concentration in their plasma remarkably decreased within the first 60 min, and then gradually declined, suggesting a two-compartment pharmacokinetic model in (V(c) 0.41+/-0.09 l/kg, V(dss) 1.7+/-0.4 l/kg, CL(tot) 10+/-2 ml/min x kg, AUC(0-4800) 2055+/-294.3 ng x min/ml). Following oral administration of aconitine to rats at two doses of 0.1 and 1.0 mg/kg b.w., the maximum plasma concentrations (C(max)) were 0.73+/-0.08 and 3.3+/-0.6 ng/ml at times of 45+/-9 and 150+/-52 min, respectively, and the AUC(0-1440) values were 130+/-4 and 1600+/-270 ng x min/ml. The bioavailability (F) of aconitine was determined to be 0.013, where only 1.3% of the aconitine administered orally was absorbed into the body fluid. PMID:12951473

Tazawa, Takako; Zhao, Huai-Qing; Li, Yan; Meselhy, Meselhy Ragab; Nakamura, Norio; Akao, Teruaki; Hattori, Masao

2003-09-01

177

Determination of blood sirolimus concentrations in liver and kidney transplant recipients using the Innofluor® fluorescence polarization immunoassay: Comparison with the microparticle enzyme immunoassay and high-performance liquid chromatography-ultraviolet method  

PubMed Central

Background Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx® analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification. Methods Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor® Certican® FPIA (Seradyn Inc.) run on a TDx® analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx® analyser (Abbott Laboratories), and HPLC (UV detection) methods. Results The Innofluor® FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%–13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r?0.900) than in the kidney transplant samples (r?0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC. Conclusion The Innofluor® FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus. PMID:19242874

Bouzas, Lorena; Hermida, Jesús

2009-01-01

178

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

PubMed Central

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

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

1992-01-01

179

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

PubMed

We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05-20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin. PMID:1737052

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

1992-02-01

180

Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance  

PubMed Central

Background In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial. Methods We adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (?6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry. Results Mean intra-assay (n?=?157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n?=?33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at ?80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose. Conclusions Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin. PMID:23056434

Martin, Richard M.; Patel, Rita; Zinovik, Alexander; Kramer, Michael S.; Oken, Emily; Vilchuck, Konstantin; Bogdanovich, Natalia; Sergeichick, Natalia; Gunnarsson, Robert; Grufman, Lisa; Foo, Ying; Gusina, Nina

2012-01-01

181

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

2013-06-01

182

Immunoassay Animations  

NSDL National Science Digital Library

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

Chung, Kyn Wai

183

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

184

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

PubMed

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

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

2014-05-01

185

Development and evaluation of an ELISA method for the determination of lipoprotein lipase mass concentration--comparison with a commercial, one-step enzyme immunoassay.  

PubMed

We developed a non-competitive, enzyme-linked, immunosorbent assay (ELISA) for the quantitation of lipoprotein lipase (LPL) in human postheparin plasma using affinity-purified antihuman milk lipoprotein lipase antibodies produced in chicken eggs and a monoclonal antibody directed against human lipoprotein lipase. We compared our ELISA method with a commercially available sandwich-enzyme immunoassay (Markit-F LPL EIA Kit, Dainippon Pharmaceutical Co, Ltd. Osaka, Japan). The reference values for lipoprotein lipase catalytic activity concentration and mass concentration in healthy Finns were determined. Lipoprotein lipase activity concentration (mean +/- SD) was 297 +/- 112 U/l in women, and mass concentration as measured by the ELISA method was 1058 +/- 367 micrograms/l. The corresponding values for men were 247 +/- 97 U/l and 815 +/- 207 micrograms/l, respectively. Across the whole concentration range of the ELISA method, the control samples' intra- and inter-assay coefficients of variation (CV) were 5.1% and 6.5%, respectively. The correlation between the ELISA and EIA methods was good, r = +0.81. The importance of the correct standardisation of immunoassays is discussed. PMID:8864403

Antikainen, M; Suurinkeroinen, L; Jauhiainen, M; Ehnholm, C; Taskinen, M R

1996-07-01

186

Determination of polynuclear aromatic hydrocarbons (PAHs) in soil and water by a magnetic particle-based enzyme immunoassay system  

SciTech Connect

Use of immunoassays as field-screening methods to detect environmental contaminants has increased dramatically in recent years. Immunochemical assays are sensitive, rapid, reliable, cost-effective and can be used for lab or field analysis. A magnetic particle-based immunoassay system has been developed for the quantitation of polynuclear aromatic hydrocarbons (PAHs) in soil and water. Paramagnetic particles used as the solid-phase allow for the precise addition of antibody and nondiffusion limited reaction kinetics. The magnetic particle-based immunoassay is ideally suited for on-site investigation and remediation processes to delineate PAH contamination. This system includes easy-to-use materials for collection, extraction, filtration and dilution of soil samples prior to analysis by immunoassay. When analyzing water samples, a simple dilution of the sample with methanol is performed during sample collection. The method detects PAHs, including anthracene, chrysene, fluoranthene, phenanthrene, pyrene and benzo[a]pyrene, at sub-parts-per-million levels in soil and at less than 1 ppb in water. The typical precision of the assay (within assay) in soil and water is less than 15% and 12%, respectively. Recovery studies (based on phenanthrene) from soil averaged 108%, and 107% from water. The analysis of soil samples by this ELISA correlate well with Method 8310, yielding a correlation coefficient (r) of 0.963; when water samples were compared to method 8270, a regression (r) of 0.987 was obtained. The application of this ELISA method permits the cost-effective evaluation of samples with minimal solvent disposal and can result in savings of time and money. The system`s flexibility allows the analysis of PAHs in many other sample matrices with minimum sample preparation.

Rubio, F.M.; Lawruk, T.S.; Lachman, C.E.; Herzog, D.P. [Ohmicron Environmental Diagnostics, Newtown, PA (United States); Fleeker, J.R. [North Dakota State Univ., Fargo, ND (United States)

1995-12-31

187

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

PubMed

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

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

2014-05-20

188

Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection. An analytical comparison.  

PubMed

Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60-90% range by ELISA and in the 50-90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation. PMID:10074694

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

1999-02-12

189

Immunoassay for Simazine and Atrazine with Low Cross-Reactivity for Propazine  

E-print Network

Immunoassay for Simazine and Atrazine with Low Cross-Reactivity for Propazine Monika Wortberg: Immunoassay; enzyme-linked immunosorbent assay (ELISA); simazine; atrazine; propazine; triazine herbicide pesticide contami- nation, several immunoassays for atrazine have been developed as screening tools

Hammock, Bruce D.

190

Serum IgM antibodies contribute to high levels of opsonophagocytic activities in toddlers immunized with a single dose of the 9-valent pneumococcal conjugate vaccine.  

PubMed

In immunogenicity trials of pneumococcal conjugate vaccines (PCVs), only IgG antibody concentrations to pneumococcal capsular polysaccharides (PPSs) are usually determined, along with the opsonophagocytic activity (OPA) of antipneumococcal antibodies. We aimed to determine the role of both IgG and IgM in OPA in toddlers receiving one dose of 9-valent PCV (PCV9). The IgG and IgM antibody concentrations to PPSs of serotypes 6A, 9V, 14, 19F, and 23F were measured by enzyme immunoassay in sera from toddlers (ages 18 to 35 months) 1 month after a single PCV9 dose. The OPA for the same serotypes was measured by multiplexed opsonophagocytosis assay (MOPA). Further, IgG and IgM concentrations and MOPA were measured to PPS of serotypes 6A, 14, and 19F in sera collected 12 months after vaccination. The detected MOPA titers were high in comparison to the IgG concentrations 1 month after immunization. The IgM concentrations were higher than IgG concentrations for serotypes 6A and 14 (P < 0.001) and as high as IgG for serotypes 9V, 19F, and 23F. Correlation of the IgM antibody concentrations with MOPA (r = 0.35 to 0.65) was stronger compared to that of the IgG antibodies (r = 0.07 to 0.41). The depletion of IgG antibodies in three sets of pooled sera only slightly decreased the OPA activity against serotype 14. At 12 months after immunization, 50 to 100% of serum samples still showed detectable MOPA activity against serotypes 6A, 14, and 19F. Our results suggest that IgM contributes to OPA 1 month after a single PCV9 vaccination in toddlers and that functionally active IgM and IgG antibodies persist for at least a year. PMID:22875604

Simell, Birgit; Nurkka, Anu; Ekström, Nina; Givon-Lavi, Noga; Käyhty, Helena; Dagan, Ron

2012-10-01

191

Anti-bovine IgM monoclonal antibodies produced by hybrid cells after in vitro immunization as detected by enzyme-linked immunosorbent assay  

E-print Network

-bovine IgG 1: 1 Mouse anti-bovine IgG 1:5 Culture media 1. 095 0. 792 0. 063 ~Anti en Bovine IgG at 200 ng/we11 17 Optical density at 280 nm 2. 000 1, 000 . 699 . 523 . 397 . 301 . 222 . 155 . 100 . 046 Fraction; 65 60 55 50 45 40 35 30... 25 20 15 10 5 Figure 2: Chromatographic separation of euglobulin fraction of bovine serum. 18 1. Fractions 1-13 2. 14-20 21-34 35-43 O 0? 5. 44-56 6. Whole bovine serum control A. Mouse anti-bovine IgM Figure 3: Agar gel immunodiffusion...

Hunter, Doris Marie

1983-01-01

192

Detection of West Nile Virus Antigen in Mosquitoes and Avian Tissues by a Monoclonal Antibody-Based Capture Enzyme Immunoassay  

PubMed Central

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 102.1 to 103.7 PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 103 PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay. PMID:12037058

Hunt, Ann R.; Hall, Roy A.; Kerst, Amy J.; Nasci, Roger S.; Savage, Harry M.; Panella, Nicholas A.; Gottfried, Kristy L.; Burkhalter, Kristen L.; Roehrig, John T.

2002-01-01

193

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

194

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

SciTech Connect

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

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

2006-06-16

195

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

PubMed

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

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

2011-01-01

196

Autoantibodies against the exocrine pancreas in autoimmune pancreatitis: gene and protein expression profiling and immunoassays identify pancreatic enzymes as a major target of the inflammatory process  

PubMed Central

Objectives Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. Methods In order to explore key targets of the inflammatory process we analysed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, Western blot and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared to those of 8 patients with non-AIP chronic pancreatitis (CP). Results Expression profiling revealed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry demonstrated a near-loss of trypsin positive acinar cells, which was also confirmed by Western blotting. The serum of AIP patients contained high titres of autoantibodies against the trypsinogens PRSS1, and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals we found similar protein patterns and a reduction in trypsinogen. Conclusion These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP. PMID:20407433

Löhr, J.-Matthias; Faissner, Ralf; Koczan, Dirk; Bewerunge, Peter; Bassi, Claudio; Brors, Benedikt; Eils, Roland; Frulloni, Luca; Funk, Anette; Halangk, Walter; Jesenofsky, Ralf; Kaderali, Lars; Kleeff, Jörg; Krüger, Burkhard; Lerch, Markus M.; Lösel, Ralf; Magnani, Mauro; Neumaier, Michael; Nittka, Stephanie; Sahin-Tóth, Miklós; Sänger, Julian; Serafini, Sonja; Schnölzer, Martina; Thierse, Hermann-Josef; Wandschneider, Silke; Zamboni, Giuseppe; Klöppel, Günter

2011-01-01

197

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.

1996-01-01

198

[Evaluation of new fourth-generation human immunodeficiency virus antigen and antibody detection assay with enzyme-linked fluorescent immunoassay].  

PubMed

We evaluated the fourth-generation HIV screening assay VIDAS HIV DUOII (DUOII) based on ELFA for simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies and HIV-1 p24 antigen through comparison with other HIV antigen-antibody detection assays. Materials were 1228 HIV-negative specimens, 95 HIV-antibody-positive specimens, and HIV commercial panels. The specificity of DUOII was 99.8% and sensitivity 100%, detecting all of HIV-1 group M subtype A, B, B', C, D, A/E, F, G, B/D, HIV-1 group O, and HIV-2. The sensitivity test to HIV-1 p24 antigen was 5pg/ mL, higher than other assays. DUOII was equivalent to or superior in detecting results earlier than other assays in an evaluation using 10 commercial HIV-1 seroconversion panels of primary infection. DUOII detects anti-HIV IgM antibody, so no negative sample was found in the second window between p24 antigen disappearance and raised anti-HIV IgG antibody. DUOII has sufficient specificity and sensitivity for HIV screening, and detects primary infection sooner than other assays. These results indicate that DUOII is useful and reliable in HIV screening. PMID:17966638

Shima, Takako; Sudo, Koji; Kondo, Makiko; Kurai, Hanako; Sagara, Hiroko; Imai, Mitsunobu

2007-09-01

199

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

PubMed

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

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

2014-09-01

200

Is a Two-Step Glutamate Dehyrogenase Antigen-Cytotoxicity Neutralization Assay Algorithm Superior to the Premier Toxin A and B Enzyme Immunoassay for Laboratory Detection of Clostridium difficile?  

Microsoft Academic Search

A two-step algorithm for the detection of Clostridium difficile by the use of C. Diff Quik Chek (TechLab, Blacksburg, VA) and a tissue culture cytotoxicity neutralization assay was found to be more sensitive than the widely used solid-phase enzyme immunoassay (EIA), the Premier toxin A and B EIA (Meridian Bioscience, Cincinnati, OH), and a newly developed, rapid single-test EIA for

Peter H. Gilligan

2008-01-01

201

The clinical significance of measuring different anti-dsDNA antibodies by using the Farr assay, an enzyme immunoassay and a Crithidia luciliae immunofluorescence test.  

PubMed

Anti-double-stranded DNA (dsDNA) antibodies are highly specific for the diagnosis of systemic lupus erythematosus (SLE) but are heterogeneous in respect to, for example, avidity, class and cross-reactivity. Sera from 2061 patients were measured by three methods: an enzyme-linked immunosorbent assay (ELISA), an indirect immunofluorescence test with Crithidia luciliae as substrate (CLIF), and the Farr assay, a radioimmunological method based on the ammonium sulfate precipitation of immune complexes. The different anti-dsDNA antibody determinations were evaluated by analysis of patient records. The reason for a reactive Farr assay in 14 patients was predominantly the measurement of antibodies of the IgM class, which are not detected by the ELISA. The detection of additional antibodies to dsDNA of the IgA class, to single-stranded DNA or to histones plays a minor role. In comparison with the Farr assay, we found more positive results with the ELISA, which additionally detects anti-dsDNA antibodies of low avidity. The ELISA might also yield positive values in conditions such as chronic liver diseases, various infections and connective tissue diseases other than SLE. Avoiding the disadvantages of radioactivity, the ELISA is well suited as a screening test for dsDNA antibodies. However, positive results should be confirmed by the CLIF test or preferably by the Farr assay, thus combining sensitivity with specificity. PMID:1304405

Werle, E; Blazek, M; Fiehn, W

1992-12-01

202

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

203

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

PubMed

Several methods for the rapid and specific detection of Salmonella spp. in meat have been described. This study was conducted to evaluate the capability of the VIDAS(®) UP (SPT [Salmonella Phage Technology]), an enzyme-linked fluorescent immunoassay method, and fluorescence in situ hybridization (FISH) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella spp. from beef, pork, and poultry meat samples. The meat was inoculated with a mixture of Salmonella spp. on three levels of contamination. It was also checked that the tests did not produce cross-reactions with other Enterobacteriaceae rods. On the basis of the results, the relative specificity, relative accordance, and relative sensitivity of the method were determined. In meat samples, Vidas UP and FISH detection results were in substantial agreement with ISO, with relative specificity, accordance, and sensitivity rates of 90%, 96.3%, and 100%, respectively, for Vidas UP and 100%, 100%, and 99.4%, respectively, for FISH. This is the first report on the evaluation of both Vidas UP and FISH compared to ISO for the rapid detection of Salmonella enterica serovars in meat. PMID:24971928

Zadernowska, Anna; Chaj?cka-Wierzchowska, Wioleta; K??bukowska, Lucyna

2014-09-01

204

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

PubMed

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

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

2015-01-20

205

Utility of Galactomannan Enzyme Immunoassay and (1,3) ?-d-Glucan in Diagnosis of Invasive Fungal Infections: Low Sensitivity for Aspergillus fumigatus Infection in Hematologic Malignancy Patients ?  

PubMed Central

Previous studies have reported that galactomannan (GM) enzyme immunoassay and 1,3 beta-glucan (BG) assay may be useful diagnostic tools, but their sensitivities are variable. We compared the performances of both tests. Between October 2002 and May 2005, 82 patients were prospectively monitored for 12 weeks. A total of 414 samples were tested by GM assay and 409 samples were tested by BG assay for the following four groups of patients: those with invasive aspergillosis (IA), those with other mold infections (Fusarium, scedosporium, zygomycosis, etc.), those with candidemia, and control patients. Blood samples were obtained twice on week 1 and once every other week for a total of 12 weeks. Patients in the invasive fungal infection groups had comparable risk factors. The sensitivity of the GM test was significantly higher for patients with IA due to non-fumigatus Aspergillus species than for patients with IA due to Aspergillus fumigatus (49% versus 13%; P < 0.0001) or with other mold infections (49% versus 6%; P < 0.0001). However, the sensitivity range (47% to 64%) and specificity (88%) of the BG assay were comparable among all patients tested, regardless of the infecting pathogen. The performance of GM-based diagnosis appears to be better for detecting non-fumigatus Aspergillus species. The diagnostic marker BG was shown to have a higher sensitivity than that of GM in detecting IA and other mold infections in hematologic malignancy patients. PMID:19005145

Hachem, R. Y.; Kontoyiannis, D. P.; Chemaly, R. F.; Jiang, Y.; Reitzel, R.; Raad, I.

2009-01-01

206

A method for rating sugarcane cultivars for resistance to ratoon stunting disease based on an enzyme-linked immunoassay  

Microsoft Academic Search

The evaporative-binding enzyme-linked immunosorbent assay (EB-EIA) was used to compare the relative populations of Leifsonia (Clavibacter) xyli subsp. xyli, the causal agent of ratoon stunting disease, in 25 cultivars of sugarcane that were inoculated by planting them through\\u000a a contaminated mechanical planter. There were highly significant differences between cultivars in relative populations of\\u000a bacteria in xylem extracts. However, the percentage

Barry J. Croft

2002-01-01

207

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

PubMed

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

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

2014-01-01

208

Use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening enzyme immunoassay results for the serological diagnosis of syphilis.  

PubMed

We studied the use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening results of the Murex ICE Syphilis enzyme immunoassay (EIA) with the confirmatory results of both the Serodia Treponema pallidum particle agglutination (TPPA) and the fluorescent treponemal antibody-absorption (FTA-Abs) assays, for the serological diagnosis of syphilis. This was an observational study on the serum samples received by the Syphilis Laboratory, Hong Kong, during the period from January 2006 to December 2012. A total of 801 serum samples with discordant positive screening EIA results were used. Consensus results of such serum samples were derived from results of the EIA, TPPA and FTA-abs assays. The age range of the individuals was 14 to 104 years (median of 52). There were 369 males and 432 females. Of 378 serum samples, 139 showed agreement among positive results, 23 of 310 showed agreement among indeterminate results and 277 of 465 showed agreement among negative results. The proportions of agreement among positive, indeterminate and negative results were 0.37 (95% CI 0.32-0.42), 0.07 (95% CI 0.05-0.11) and 0.60 (95% CI 0.55-0.64), respectively; kappa 0.55 (95% CI 0.49-0.60). There were 60 serum samples with positive consensus results but negative INNO-LIA syphilis score results and 10 with negative consensus results but positive INNO-LIA syphilis score results. Although the INNO-LIA syphilis score assay can be considered a valid alternative confirmatory test for the serological diagnosis of syphilis, the present study showed that its use in the resolution of discordant positive screening EIA results was moderate. A more extensive characterization of serum samples with discordant reactive screening treponemal test results is necessary. PMID:23970631

Lam, T K; Lau, H Y; Lee, Y P; Fung, S M; Leung, W L; Kam, K M

2014-01-01

209

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

PubMed

Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11?-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11?-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11?-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11?-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species. PMID:25066418

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

2014-09-15

210

Validation of an enzyme immunoassay for assessing adrenocortical activity and evaluation of factors that affect levels of fecal glucocorticoid metabolites in two New World primates.  

PubMed

Non-invasive methods to assess stress hormone output via fecal glucocorticoid metabolites (FGCMs) have become a powerful tool in behavioral studies and conservation biology because they allow exploring the link between behavior, an animal's socio-ecological environment and its adrenocortical activity. However, FGCM levels are influenced by numerous other factors which often confound their interpretation. Thus, before applying these methods, knowledge on the impact of these factors is important. In this study we investigated the effect of (1) time of day, (2) age, (3) sex and (4) female reproductive state on FGCM levels in brown spider monkeys (Ateles hybridus) and red howler monkeys (Alouatta seniculus). Initially, we validated a 11?-hydroxyetiocholanolone enzyme immunoassay for monitoring the physiological stress response via fecal analysis in both species. We determined FGCM levels in fecal samples collected from two and six groups of wild spider monkeys (n=461 samples) and howler monkeys (n=166 samples), respectively. Our analyses revealed a strong effect of time of day on FGCM levels in spider monkeys, but no effect in howler monkeys. Adults of both species had significantly higher FGCM levels than subadults. In neither of the two species we found a sex-effect on FGCM output. Reproductive condition strongly affected FGCM levels in female spider monkeys which showed increasing concentrations with progressing gestation. This was not investigated in female howler monkeys due to an insufficient sample size. Our data indicate that the influence of the tested factors on fecal glucocorticoid metabolite output is species-specific, and that these variables need to be considered when interpreting FGCM levels in the species. PMID:23707497

Rimbach, Rebecca; Heymann, Eckhard W; Link, Andrés; Heistermann, Michael

2013-09-15

211

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

212

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

213

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

PubMed

Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology. PMID:19335228

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

2009-01-01

214

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

PubMed

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

Kajaysri, Jatuporn; Nokkaew, Weerapun

2014-03-01

215

Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples.  

PubMed

VIDAS Salmonella (VIDAS-SLM) is an automated system that uses the enzyme-linked fluorescent assay method to detect Salmonella species. This study evaluated the efficacy of the VIDAS-SLM method in detecting Salmonella species in pork carcass sponge samples gathered from 10 slaughter plants in Taiwan. Two hundred fifty-seven pork carcass sponge samples were screened by the VIDAS-SLM method and by the culture method in parallel. While 18 sponge samples were found to test positive by both methods, the VIDAS-SLM method detected four additional positive samples for which the culture method failed to recover Salmonella. The specificity of the VIDAS-SLM method was found to be 0.98, and its sensitivity was 1.0, since no false-negative results occurred. Artificially inoculated Salmonella at concentrations as low as 5.0 x 10(0) CFU/ml was detected in the heat-inactivated sponge sample in the presence or absence of 5.0 x 10(4) CFU of Citrobacter freundii per ml. Thus, the VIDAS-SLM method is a rapid screening method and a potential alternative to the time- and labor-intensive culture method. PMID:12380756

Yeh, Kuang-Sheng; Tsai, Chin-En; Chen, Shih-Ping; Liao, Chao-Wei

2002-10-01

216

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.  

PubMed

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

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

2014-01-01

217

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

PubMed Central

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

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

2014-01-01

218

Diagnosis of invasive candidiasis by detection of mannan antigen by using the avidin-biotin enzyme immunoassay.  

PubMed Central

The diagnosis of invasive candidiasis was attempted by detection of circulating mannan antigen by using an avidin-biotin-amplified enzyme-linked immunosorbent assay (AB-ELISA), and this method was compared with the conventional culture method. Mannan antigen was detected by AB-ELISA in the sera of 16 (84.2%) of the 19 patients with invasive candidiasis. On the other hand, for 34 immunocompromised candidiasis-free patients, including 8 with aspergillosis or cryptococcosis, mannan antigen was positive during only 1 of the 67 febrile episodes and in the serum of none of the 50 outpatients without infections. The results were also negative for all patients with deep-seated mycoses other than candidiasis. However, the mannan level was low (less than 2.0 ng/ml) in the serum of 63.2% of the patients with invasive candidiasis. The positivity rate of blood cultures was 31.6%, and that of blood cultures and/or cultures of samples from sterile sites combined was 47.4%. The advantages of the diagnosis based on antigen detection by AB-ELISA are considered to be a higher sensitivity and elimination of nonspecific reactions by the introduction of the avidin-biotin system and pretreatment of sera by heating. In addition, it is considered essential for high sensitivity that transient mannan antigenemia be determined frequently so that it is not overlooked. In light of its sensitivity and specificity, this method is considered to be clinically useful in the diagnosis of invasive candidiasis. PMID:1774238

Nakamura, A; Ishikawa, N; Suzuki, H

1991-01-01

219

Microfluidic Immunoassays  

Microsoft Academic Search

Immunoassays have long been widely used in a variety of applications, such as for medical diagnostics, pharmaceutical analysis, environmental, food safety testing, and for basic scientific investigations because of its simplicity, sensitivity, and specificity. Microfluidic systems, also well known as a “lab-on-a-chip” or a “micro-total-analysis-system” have attracted a lot of attention in the past two decades because of advantages associated

Chun-Che Lin; Jung-Hao Wang; Hui-Wen Wu; Gwo-Bin Lee

2010-01-01

220

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies.  

PubMed

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis. PMID:19547868

Rodrigues, I M X; Castro, A M; Gomes, M B F; Amaral, W N; Avelino, M M

2009-05-01

221

A general strategy for photoelectrochemical immunoassay using an enzyme label combined with a CdS quantum dot/TiO? nanoparticle composite electrode.  

PubMed

Photoelectrochemical (PEC) immunoassay has received increasing attention owing to its good analytical performance and attractive potential for future protein assay. This Letter represents a novel and general strategy for elegant PEC immunoassay of the important cardiac marker troponin T (cTnT) at neutral conditions. Specifically, we first developed an efficient CdS quantum dots (QDs)/TiO2 nanoparticles (NPs) photoelectrode, on the basis of which an exquisite ?-galactosidase (?-Gal) catalytic system was integrated with sandwich immunobinding for probing cTnT. In pH 7.4, ?-Gal could catalyze the conversion of p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP), which could be easily photo-oxidized to p-quinone imine (PQI). Because the resulting photocurrent was directly related with the target concentration, an innovative PEC immunoassay could be realized for cTnT detection. The neutral operating condition of this protocol would greatly contribute to its wide applicability for protein assay. This work provides the first PEC immunoassay toward cardiac marker and, more significantly, opens a different perspective for future PEC immunoassay development through a general sensing protocol. PMID:25403364

Zhao, Wei-Wei; Chen, Ru; Dai, Pan-Pan; Li, Xiang-Ling; Xu, Jing-Juan; Chen, Hong-Yuan

2014-12-01

222

A water soluble antigen unique to Brucella abortus Strain 19: isolation, immunological characterization and use in an enzyme immunoassay for differential diagnosis of vaccinated and Brucella abortus infected cattle  

E-print Network

A WATER SOLUBLE ANTIGEN UNIQUE TO BRUCELLA ABORTUS STRAIN 19: ISOLATION, IMMUNOLOGICAL CHARACTERIZATION AND USE IN AN ENZYME IMMUNOASSAY FOR DIFFERENTIAL DIAGNOSIS OF VACCINATED AND BRUCELLA ABORTUS INFECTED CATTLE A Thesis by CHARLES RIPLEY...':1. 1. A A BOB'I'US STRAIN 19: ISOLATION, IMMUiVOI. OGIC. & I, CH!& IIACTLIIIZATIi&N AND I. 'Sl IN AN ENZY&IL' IIVIMUNOASSAY I'OR Dll'I'EIIEiNTLAL DIAGNOSIS OE VACCINATED AVD BRL!CELLA ABORTUS 1NI" ECTED CATTf. L' A T!icsis h& CIIARLES Rl!'l, l...

Smithwick, Charles Ripley

1983-01-01

223

Is a two-step glutamate dehyrogenase antigen-cytotoxicity neutralization assay algorithm superior to the premier toxin A and B enzyme immunoassay for laboratory detection of Clostridium difficile?  

PubMed

A two-step algorithm for the detection of Clostridium difficile by the use of C. Diff Quik Chek (TechLab, Blacksburg, VA) and a tissue culture cytotoxicity neutralization assay was found to be more sensitive than the widely used solid-phase enzyme immunoassay (EIA), the Premier toxin A and B EIA (Meridian Bioscience, Cincinnati, OH), and a newly developed, rapid single-test EIA for C. difficile toxins A and B (Tox A/B Quik Chek; TechLab). PMID:18256226

Gilligan, Peter H

2008-04-01

224

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

225

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

PubMed Central

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

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

1991-01-01

226

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera.  

PubMed

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered. PMID:2613705

Thraenhart, O; Ramakrishnan, K

1989-10-01

227

Immunoassays for pesticide monitoring  

NASA Astrophysics Data System (ADS)

This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.

Wengatz, Ingrid; Szurdoki, Ferenc; Swamy, Anand R.; Evans, Lawrence, III; Patonay, Gabor; Stimmann, Eric; Delwiche, Michael; Stoutamire, Donald; Gee, Shirley J.; Hammock, Bruce D.

1995-05-01

228

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

229

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

230

Enzyme  

MedlinePLUS

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

231

Purification, characterization of O-acetylated sialoglycoconjugates- specific IgM, and development of an enzyme-linked immunosorbent assay for diagnosis and follow-up of Indian visceral leishmaniasis patients  

Microsoft Academic Search

The surface expression of 9-O-acetylated sialic acid (9-OAcSA) is elevated on hematopoietic cells and erythrocytes of visceral leishmaniasis (VL) patients. In this study, we show that VL patients contain elevated levels of IgM antibodies directed against 9- O- acetylated sialoglycoconjugates (9-OAcSG). These antibodies were affinity purified with bovine submaxillary protein as the affinity matrix containing the terminal epitope, 9-OAcSA2-6GalNAc. They

Sumi Bandyopadhyaya; Mitali Chatterjee; Santanu Pal; Ross F. Waller; Shyam Sundar; Malcolm J. McConville; Chitra Mandala

232

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

NASA Astrophysics Data System (ADS)

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

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

2008-10-01

233

Colorimetric Immunoassay for Detection of Tumor Markers  

PubMed Central

Tumor markers are substances, usually proteins, produced by the body in response to cancer growth, or by the cancer tissue itself. They can be detected in blood, urine, or tissue samples, and the discovery and detection of tumor markers may provide earlier diagnosis of cancer and improved therapeutic intervention. Colorimetric immunoassays for tumor marker detection have attracted considerable attention, due to their simplicity and high efficiency. The traditionally used colorimetric immunoassays for the detection of tumor markers are based on enzyme-linked immunosorbent assays, and the great achievement of nanotechnology has further opened opportunities for the development of such kind of immunoassays. This paper will summarize recent advances in the field of colorimetric immunoassays for detecting tumor markers, which is aimed to provide an overview in this field, as well as experimental guidance for the learner. PMID:21614193

Yin, Yongmei; Cao, Ya; Xu, Yuanyuan; Li, Genxi

2010-01-01

234

Positive predictive value of anti-HCMV IgM as an index of primary infection.  

PubMed

A search for specific IgM antibodies was used for the detection of primary human cytomegalovirus (HCMV) infections, but the significance of the results is limited by the possible persistence of specific IgM over time, the fact that they are also produced during episodes of reactivation or reinfection, and possible cross-reactions with other viruses. Anti-HCMV antibody screening was carried out to assess the positive predictive value of detecting specific IgM antibodies using IgG and IgM enzyme-linked immunosorbent assays (ELISAs) in 6990 patients examined during the period 2005-2007. In comparison with IgG avidity, the positive predictive value of screening by IgM ELISA alone was 49.3%, which increased to 73% when the presence of IgM was confirmed by an enzyme-linked fluorescent assay (ELFA). The predictive values of highly or weakly positive IgM ELISA alone were respectively 68.8% and 16.4%, but increased to 83.1% and 39.1% if IgM was confirmed by ELFA. The positive predictive value of the IgM/IgG ratio ranged from 26.7% for a low ratio and the detection of IgM by ELISA alone, to 90.7% for a high ratio and ELFA-confirmed IgM detection. These findings indicate that a specimen in which highly positive IgM ELISA values are confirmed by ELFA, or which shows a high IgM/IgG ratio, is a good indicator of infection occurring within the previous three months. PMID:20470827

De Paschale, Massimo; Agrappi, Carlo; Manco, Maria Teresa; Clerici, Pierangelo

2010-09-01

235

Identification of a novel host-specific IgM protease in Streptococcus suis.  

PubMed

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

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

2013-03-01

236

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

237

Kinase Activity Studied in Living Cells Using an Immunoassay  

ERIC Educational Resources Information Center

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

Bavec, Aljos?a

2014-01-01

238

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

E-print Network

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

Ball, Madalyn Eunice

1993-01-01

239

Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay.  

PubMed

Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 ?M) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats. PMID:25586260

Hu, Dongmei; Fry, Scott R; Huang, Johnny X; Ding, Xixia; Qiu, Liwen; Pan, Yuxian; Chen, Yue; Jin, Jing; McElnea, Catriona; Buechler, Joe; Che, Xiaoyan; Cooper, Matthew A

2013-01-01

240

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. PMID:22539474

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

2012-01-01

241

Dust in the IGM: pro and contra  

NASA Astrophysics Data System (ADS)

Observations of dust grains in the intergalactic medium (IGM) allow us to study an important aspect in the evolution of galaxies. Although its existence had been previously speculated upon, direct evidence of the presence of dust in the intergalactic space has only been available recently. We discuss various issues regarding the presence of dust in the IGM—its sources, transport mechanisms from galaxies into the IGM, its effect on reddening and on cosmological studies.

Shchekinov, Y. A.; Nath, B.

2011-09-01

242

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...

243

PCR of DNA from dried blood spots on filter paper coupled to a simple DNA enzyme immunoassay for rapid detection of HIV1  

Microsoft Academic Search

Conclusions The application of PCR and techniques confirming the specificity of PCR products in routine diagnostic laboratories, requires that the procedures are simple and reproducible. The microtitre plate assay we described for detection of HIV-1 is sensitive, simple, rapid and reproducible. The DEIA test is perfectly compatible with standard enzyme linked immunosorbent assay equipment, permitting the processing of a large

B. Schweiger; C. Kücherer; C. Fleischer; H. v. Spreckelsen; P. Zablocki-Kaiser; G. Pauli

1996-01-01

244

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

245

PAPER www.rsc.org/loc | Lab on a Chip Microfluidic device for immunoassays based on surface plasmon resonance  

E-print Network

PAPER www.rsc.org/loc | Lab on a Chip Microfluidic device for immunoassays based on surface plasmon to about 60 min. Introduction Immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), have-step immunoassays. This feature limits their applications, especially when the detection method is sensitive

Zare, Richard N.

246

Serodiagnosis of Lyme borreliosis using detection of different immunoglobulin (sub)classes by enzyme-linked immunosorbent assay and Western blotting.  

PubMed

To improve the performance of enzyme-linked immunosorbent assays for the serodiagnosis of Lyme borreliosis, the prevalence of several immunoglobulin classes and subclasses against various antigens of Borrelia burgdorferi was investigated by Western blotting. The sera of 40 early Lyme borreliosis patients (ELB), 27 late Lyme borreliosis patients (LLB), 62 healthy controls and 140 non-Lyme borreliosis patients were used. Detection of IgG1 versus total IgG was found to be more sensitive in detecting Borrelia burgdorferi antigens, especially flagellin (41 kD) protein, but did not improve the performance of Western blotting. The use of IgG1 detection showed an increase in sensitivity and specificity for the early Lyme borreliosis patient group compared to the standard IgG and IgM detection method by enzyme immunoassays using purified Borrelia burgdorferi flagellum. However, in an enzyme immunoassay using a total sonicate, sensitivity in detecting early Lyme borreliosis and late Lyme borreliosis with IgG1 remained lower compared to the detection of early Lyme borreliosis by IgM antibodies and late Lyme borreliosis by total IgG antibodies. PMID:11214222

Goossens, H A; van den Bogaard, A E; Nohlmans, M K

2001-01-01

247

Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C. difficile Toxin Enzyme Immunoassays with C. difficile Nucleic Acid Amplification Testing Increase Diagnostic Yield in a Tertiary Pediatric Population  

PubMed Central

We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens. PMID:22259201

McGowan, Karin L.

2012-01-01

248

A New Efficient Method for Eliminating the Interference Effect of Human Serum and Increasing the Sensitivity and Recovery Rate of Enzyme Immunoassay  

Microsoft Academic Search

A new, very simple method for increasing the sensitivity and recovery rate of enzyme?linked immunosorbent assay (ELISA) for the precise quantification of antigen in human serum is described. The assay design uses CATNF6A4c IgG2a monoclonal antibody and biotinylated anti?human tumor necrosis factor?? (hTNF??) polyclonal mouse IgG as the capture and tracer antibodies, respectively. The assay is completed within 4 hours

Resul Karakus; Bugra A. Buyrukcu; Cemalettin Aybay

2005-01-01

249

Plasma Levels of ADAMTS13 Antigen Determined with an Enzyme Immunoassay Using a Neutralizing Monoclonal Antibody Parallel ADAMTS13 Activity Levels  

Microsoft Academic Search

Measurements of plasma ADAMTS13 activity (ADAMTS13: AC) have been used for the diagnosis of patients with thrombotic thrombocytopenic\\u000a purpura (TTP); however, the clinical usefulness of plasma ADAMTS13 antigen (ADAMTS13:AG) has been controversial, because antigen\\u000a values vary widely among patients with acquired idiopathic TTP (ai-TTP). We have developed a novel enzyme-linked immunosorbent\\u000a assay (ELISA) for the determination of plasma ADAMTS13:AG. This

Hideo Yagi; Shin Ito; Seiji Kato; Hisahide Hiura; Masanori Matsumoto; Yoshihiro Fujimura

2007-01-01

250

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

PubMed

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

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

1994-12-01

251

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

252

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

253

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. PMID:16339069

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

2005-01-01

254

Comparison of Premier CAMPY Enzyme Immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY Tests with Culture for Laboratory Diagnosis of Campylobacter Enteric Infections ? †  

PubMed Central

Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the “gold standard” for diagnosis. PMID:20810765

Granato, Paul A.; Chen, Li; Holiday, Iris; Rawling, Russell A.; Novak-Weekley, Susan M.; Quinlan, Tammy; Musser, Kimberlee A.

2010-01-01

255

Evaluation of a multiplex flow immunoassay for detection of epstein-barr virus-specific antibodies.  

PubMed

Conventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immunofluorescence 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 IgM class antibodies to the EBV viral capsid antigen (VCA) and IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1). Serum specimens (n = 1,315) submitted for routine EBV-specific antibody testing by EIA (Grifols-Quest, Inc., Miami, FL) were also tested by the multiplex bead assay using the BioPlex 2200 automated analyzer. Specimens showing discordant results were tested by IFA. Following IFA resolution, the BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated 97.9%, 91.4%, and 96.9% agreement, respectively, with the results obtained by EIA. Furthermore, the BioPlex assays showed an overall agreement of 94.1% with the EIA when the specimens were categorized by disease state (susceptible, acute, or past infection) based on the EBV-specific antibody profiles. These findings indicate that the BioPlex EBV assays demonstrate a performance comparable to that of the conventional EIA, while allowing for a more rapid (2.3 h for 100 samples versus 4.5 h by the EIA) and higher-throughput ( approximately 400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response. PMID:18632919

Binnicker, M J; Jespersen, D J; Harring, J A; Rollins, L O; Beito, E M

2008-09-01

256

IgM nephropathy revisited  

PubMed Central

IgM nephropathy (IgMN) is an idiopathic immune complex-mediated glomerulopathy that was first described as a distinct disease in a nephropathology literature in 1978. Here, a historical review and the current status of IgMN in the light of world literature and the current experience will be presented. The Pubmed (www.pubmed.gov) search was made for articles on IgMN as the sole subject of the study or where it constituted a significant number of cases in a biopsy series in the world literature written in English. A total of 41 articles were found. A critical review of the literature was made. Soon after 1978, a series of reports were published mostly from the western world, but the interest in the entity did not withstand the test of time. No substantial basic medical research was carried out and the disease was largely ignored by the western researchers. More recently, a flurry of articles have appeared in the literature on the topic, mostly from tropical countries, and have renewed the interest in the entity. However, most of the current literature on IgMN is based on clinical observations, and experimental models and mechanistic studies of IgMN are lacking. There is an urgent need to develop consensus based criteria for the diagnosis of the condition, as well as, to focus the research on mechanistic studies to understand the pathogenesis of the disease better. PMID:23573499

Mubarak, Muhammed; Kazi, Javed I

2012-01-01

257

Recombinant envelope protein-based enzyme immunoassay for IgG antibodies is comparable to neutralization tests for epidemiological studies of dengue infection.  

PubMed

Dengue virus (DENV) is the most prevalent arbovirus in the world, found mainly in tropical regions. As clinical manifestations present frequently as nonspecific febrile illness, laboratory diagnosis is essential to confirm DENV infections and for epidemiological studies. Recombinant envelope (E) antigens of four serotypes of DENV were used to develop an immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA). To evaluate the IgG-ELISA, a panel of serum samples that had been tested previously by a plaque reduction neutralization test (PRNT) was investigated for the presence of anti-E antibodies against the four DENV serotypes. IgG-ELISA was found to have a sensitivity (91%) and specificity (98%) at a receiver-operating characteristic (ROC) optimized cutoff and demonstrated high performance as well as good indexes. A concordance of 97% was achieved between both assays, and only 21/704 (3%) samples were not concordant. The results of the present study demonstrate a moderate correlation between neutralizing antibody titers and IgG-ELISA values. These findings indicate that the recombinant protein-based IgG-ELISA is a suitable method for routine serodiagnosis, monitoring and seroepidemiological studies of DENV infections. PMID:23018061

Rocha, Eliseu Soares de Oliveira; Oliveira, Jaquelline Germano de; Santos, João Rodrigues dos; Rodrigues, Gisele Olinto Libânio; Figueiredo, Leandra Barcelos; Pessanha, José Eduardo Marques; Proietti, Fernando Augusto; Fonseca, Flávio Guimarães da; Bonjardim, Claudio Antonio; Ferreira, Paulo Cesar Peregrino; Kroon, Erna Geessien

2013-01-01

258

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

PubMed

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

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

2014-09-15

259

Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay  

PubMed Central

Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on ‘antibody-antigen’ interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs. PMID:24816648

Gavrilova, Elena S.; Bobrovnik, Sergey A.; Sherriff, Gordon; Myslivets, Andrey A.; Tarasov, Sergey A.; Epstein, Oleg I.

2014-01-01

260

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

PubMed

The skeletal muscle protein troponin I (TnI) has been characterized as a potential thermally stable and species-specific biomarker of mammalian muscle tissues in raw meat and meat products. This study proposed a technique for the quantification of TnI comprising protein extraction and sandwich enzyme-linked immunosorbent assay (ELISA). The technique is characterized by a TnI detection limit of 4.8ng/ml with quantifiable concentrations ranging from 8.7 to 52ng/ml. The method was shown to be suitable for detection of TnI in mammalian (beef, pork, lamb, and horse) meat but not in poultry (chicken, turkey, and duck) meat. In particular, the TnI content in beef was 0.403±0.058mg/g of wet tissue. The TnI estimations obtained for the pork and beef samples using ELISA were comparable to the proteomic analysis results. Thus, the quantitative study of TnI can be a convenient way to assess the mammalian muscle tissue content of various meat products. PMID:25777979

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

2015-07-01

261

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

PubMed Central

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

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

2013-01-01

262

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

PubMed

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

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

2013-09-01

263

The right environment for the immunoassay  

SciTech Connect

For the US Environmental Protection Agency (EPA), the first in-house research effort began in 1987, when results of an early immunoassay field study verified the technology`s potential for environmental applications. Looking at the fundamental features of immunochemical reactions from the clinical laboratories, analytical chemists realized the potential value of these methods for hazardous waste site characterization and pesticide monitoring. Immunoassays rely on the interaction between an antibody and a target analyte. For environmental purposes, enzyme immunoassays are generally used. After the target analyte binds to the antibody, an enzymatic reaction yields a colorimetric change. This change, read visually or by a spectrophotometer, indicates the concentration of the target analyte. Promising results with assays for compounds (such as paraquat and pentachlorophenol) and compound groups (such as total petroleum hydrocarbons and polychlorinated biphenyls) spurred interest among various entrepreneurs. The first target market for immunoassays was environmental engineers and field crews who needed quick answers on-site to determine the direction of further remediation efforts.

Emon, J.M. Van; Gerlach, C.L.

1995-11-01

264

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

265

Interferences in Immunoassay  

PubMed Central

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

Tate, Jill; Ward, Greg

2004-01-01

266

Interferences in immunoassay.  

PubMed

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

Tate, Jill; Ward, Greg

2004-05-01

267

False-positive results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test.  

PubMed

Although tests for detection of immunoglobulin M (IgM) toxoplasma antibodies have been reported to have a high degree of accuracy, it is well recognized by investigators in the United States and Europe that false-positive results may occur with many of these tests, at times to an alarming degree. Unfortunately, this information is not well documented in the literature. Studies on various toxoplasma IgM test kits are frequently flawed. The investigators often use reference tests which have not previously been carefully evaluated as well as sera that were not appropriate to answer the question of how often false-positive results might occur. We recently had the unique opportunity to evaluate the accuracy of the Platelia Toxo IgM test in 575 serum samples obtained during an outbreak of toxoplasmosis which occurred in 1995 in the Capital Regional District of British Columbia, Canada. When compared with results obtained in a reference IgM enzyme-linked immunosorbent assay (ELISA), the Platelia Toxo IgM test had a sensitivity of 99.4%, specificity of 49.2%, positive predictive value of 51.9%, negative predictive value of 99.3%, and an overall agreement of 67.0%. In an attempt to resolve discrepancies between these two tests, a serological profile (Sabin-Feldman dye test, IgA and IgE antibody tests, differential agglutination [AC/HS] test, and IgG avidity method) was performed. Of 153 serum samples that were positive in the Platelia Toxo IgM test and negative in the IgM ELISA, 71 (46.4%) were negative in the Sabin-Feldman dye test. Of the serum samples that were positive in the dye test, 77 (93.9%) had a serological profile most compatible with an infection acquired in the distant past. These results reveal high numbers of false-positive results in the Platelia Toxo IgM test and highlight the importance of appropriate evaluation of commercial tests that are currently being marked. Our results also emphasize the importance of confirmatory testing to determine whether the results of an IgM antibody test reflect the likelihood of a recently acquired infection. PMID:8968902

Liesenfeld, O; Press, C; Montoya, J G; Gill, R; Isaac-Renton, J L; Hedman, K; Remington, J S

1997-01-01

268

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

2007-12-04

269

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

2005-12-13

270

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

271

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

PubMed

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

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

2014-10-01

272

Detection of bound residues in soils by sandwich-immunoassay  

SciTech Connect

Immunoassays are useful analytical instruments for the detection of many environmental compounds. This method was not introduced for the detection of non-extractable compounds in soil. So-called ``bound residues`` consist of a soil component, e.g. humic acids and an irreversibly bound pollutant. Because of the complexity of those macromolecules conventional analytical methods in general do not work. Enzyme immunoassays, in contrast, seem to have a large potential for applications and further developments in this field. The use of antibodies with high affinity to the analytes makes a selective detection of environmental pollutants possible. With the development of an enzyme-labeled sandwich-immunoassay polycyclic aromatic hydrocarbons (PAHs) irreversibly bound to humic acids were determined for the first time.

Dosch, M.; Weller, M.G.; Niessner, R. [Technical Univ. of Munich (Germany). Institute of Hydrochemistry

1995-12-31

273

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

E-print Network

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

Hammock, Bruce D.

274

Capillary Electrophoresis-Based Immunoassays: Principles & Quantitative Applications  

PubMed Central

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

Moser, Annette C.; Hage, David S.

2013-01-01

275

Simplifying site assessment of PAH using immunoassay directed field screening  

SciTech Connect

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic and mutagenic chemicals found in combustion products and industrial waste materials. The carcinogenic nature of PAHs has led state and federal agencies to control their levels in the environment. Until recently, site assessment of PAH levels in soil was limited by the high cost and long turnaround time associated with analysis methods used in commercial analytical laboratories. The recent development of PAH immunoassays provides a rapid and cost effective alternative to the traditional analytical methods. One such immunoassay was developed as a part of the D TECH tm line of environmental detection systems. This competitive immunoassay uses anti-PAH antibodies immobilized on latex particles. Antibody binding to an enzyme labeled PAH analog is inhibited by free PAH extracted from soil samples or contained in water samples. The immunoassay detects the majority of compounds on the EPA list of 16 PAH priority pollutants below the ppm level without cross reacting with BTEX, PCBs and other similar compounds. Field samples of PAH were collected at both petrogenic and pyrolytic sites of contamination and the PAH concentrations were determined by both the immunoassay and the EPA SW-846 GC/MS method 8270. Test results correlated between the two methods and the low number of false negative and false positive results confirmed the assay`s specificity for PAH. The immunoassay provides the advantages of rapid, on site analysis, and minimum sample preparation. Use of the immunoassay results in a significant savings in the cost of analysis because the cost per assay is lower than commercial analysis and only positive samples require additional confirmation by instrumental techniques. The simple format of the immunoassay allows personnel with minimal training to reliably and accurately determine the PAH content of soils.

Mullenix, M.C.; Hudak, R.T.; Melby, J.M.; Stave, J.W. [Strategic Diagnostics Inc., Newark, DE (United States)

1995-12-31

276

Clinical Significance of 7s IgM in Monoclonal IgM Diseases  

PubMed Central

The sera from 117 patients with diseases associated with a high production of monoclonal IgM were analysed for the presence of low molecular weight (7s) IgM by using a simple thin-layer Sephadex technique. 7s IgM was found in the sera of patients with myelomata (66%), lymphomata (45%), and Waldenström's macroglobulinaemia (20%), but was absent from the sera of patients with benign monoclonal macroglobulinaemia. This technique provides a cheap and practical test which may be valuable in selecting patients with lymphomata from those with benign lesions. ImagesFIG. 1 PMID:4995405

Carter, Paul M.; Hobbs, John R.

1971-01-01

277

Streamlining Immunoassays with Immiscible Filtrations Assisted by Surface Tension  

E-print Network

are currently available including enzyme-linked immunosorbent assay (ELISA), radio- immunoassay (RIA of assay must be washed multiple times between each of these steps to ensure residual reagents (e is performed at the start of the assay (i.e., no pipetting steps are necessary during the assay). Analytes

Beebe, David J.

278

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

279

Cloning, expression and evaluation of diagnostic potential of recombinant capsid protein based IgM ELISA for chikungunya virus.  

PubMed

The resurgence of chikungunya virus in the form of unprecedented explosive epidemic with unusual clinical severity after a gap of 32 years is a point of major public health concern. Definitive diagnosis is critical in differentiating the disease, especially in dengue endemic areas. The immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is widely used for diagnosis of chikungunya infection. However IgM ELISA based on whole virus antigen is associated with biohazard risk. The present study describes the development and evaluation of recombinant capsid protein based indirect IgM antibody capture micro plate enzyme linked immunosorbent assay (ELISA) for rapid and accurate diagnosis of chikungunya infection. The gene coding for capsid protein was cloned in frame with GST tag in pET41a+ vector and expressed in E. coli followed by purification with affinity chromatography. The comparative evaluation of in-house chikungunya IgM ELISA vis-a-vis commercially available SD ELISA kit with 90 chikungunya suspected acute phase human patient serum samples revealed 97% accordance. The overall sensitivity and specificity of the reported capsid protein based IgM ELISA was 100% and 95% respectively with 96% PPV and 100% NPV. These findings clearly demonstrated the usefulness of the recombinant capsid protein based CHIKV IgM ELISA for reliable clinical diagnosis of CHIKV infection in human patient. PMID:24681089

Priya, Raj; Khan, Mohsin; Rao, M Kameswara; Parida, Manmohan

2014-07-01

280

Multiprotein Immunoassay Arrays Fabricated by Microcontact Printing  

E-print Network

Multiprotein Immunoassay Arrays Fabricated by Microcontact Printing H. D. Inerowicz,,§ S. Howell, F. After fabrication, immunoassays were successfully performed using the patterned protein microarrays. The developed immunoassays were characterized by fluorescence microscopy and scanning probe microscopy

Reifenberger, Ronald G.

281

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

282

Immunoassay in healthcare testing applications.  

PubMed

Immunoassay is used extensively for drug testing in pain management. Drug testing for the purpose of compliance monitoring is fundamentally different from forensic applications, which may rely on immunoassay screening to rapidly identify "negative" samples. In clinical settings, focus is shifted from identification of select drugs of abuse with low positivity rates to detection of a wide variety of licit and illicit compounds with expected high positivity rates. The primary drug classes of interest in this population, opioids and benzodiazepines, require special testing considerations when immunoassay is used. This review highlights the performance characteristics of immunoassay, with special emphasis on prescription drug classes and testing at the point-of-care. PMID:25750161

DePriest, Anne Z; Black, David L; Robert, Timothy A

2015-01-01

283

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

284

Protein microchips : use for immunoassay and enzymatic reactions.  

SciTech Connect

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-{mu}m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloschuk, S.; Chupeeva, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences

2000-02-15

285

An immunoassay for human transferrin.  

PubMed

The laboratory measurement of serum transferrin is a valuable adjunct in the assessment of both iron and protein nutritional status. Conventional assays based on the Fe-binding properties of this protein are tedious to perform, susceptible to Fe contamination, and require volumes of serum that can only be obtained by venous sampling. We describe in this report a two-site enzyme immunoassay (EIA) developed with the use of monoclonal antibodies. Ten microliters serum is diluted 1:20,000 before assay, reflecting a high degree of sensitivity. The variability of this EIA is comparable to conventional colorimetric assays for total iron-binding capacity (TIBC), and excellent correspondence was observed between these methods over a range in TIBC of 150-500 micrograms/dL (27-90 mumol/L). No consistent difference was observed with the EIA when performed on venous and capillary specimens obtained simultaneously. This method will facilitate the evaluation of Fe and protein status in nutritional surveys. PMID:3276135

el Guindi, M; Skikne, B S; Covell, A M; Cook, J D

1988-01-01

286

IgM nephropathy; time to act  

PubMed Central

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

Mubarak, Muhammed

2014-01-01

287

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

288

Morphological resonances for multicomponent immunoassays  

SciTech Connect

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. [Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6142 (United States); Bronk, B.V. [United States Army Edgewood Research, Development and Engineering Center, Edgewood, Maryland 21010 (United States)

1995-06-20

289

Competitive Quenching Fluorescence Immunoassay for Chlorophenols Based on  

E-print Network

Competitive Quenching Fluorescence Immunoassay for Chlorophenols Based on Laser microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay oc- curs the opportunity for miniaturization and high- throughput screening. Fluorescence immunoassays (fluoroimmu

Hammock, Bruce D.

290

IgM Contributes to Glomerular Injury in FSGS  

PubMed Central

Glomerular IgM and C3 deposits frequently accompany idiopathic FSGS and secondary glomerulosclerosis, but it is unknown whether IgM activates complement, possibly contributing to the pathogenesis of these diseases. We hypothesized that IgM natural antibody binds to neoepitopes exposed in the glomerulus after nonimmune insults, triggering activation of the complement system and further injury. We examined the effects of depleting B cells, using three different strategies, on adriamycin-induced glomerulosclerosis. First, we treated wild-type mice with an anti-murine CD20 antibody, which depletes B cells, before disease induction. Second, we evaluated adriamycin-induced glomerulosclerosis in Jh mice, a strain that lacks mature B cells. Third, we locally depleted peritoneal B cells via hypotonic shock before disease induction. All three strategies reduced deposition of IgM in the glomerulus after administration of adriamycin and attenuated the development of albuminuria. Furthermore, we found that glomerular IgM and C3 were detectable in a subset of patients with FSGS; C3 was present as an activation fragment and colocalized with glomerular IgM, suggesting that glomerular IgM may have bound a cognate ligand. Taken together, these results suggest that IgM activates the complement system within the glomerulus in an animal model of glomerulosclerosis. Strategies that reduce IgM natural antibody or that prevent complement activation may slow the progression of glomerulosclerosis. PMID:23393315

Strassheim, Derek; Renner, Brandon; Panzer, Sarah; Fuquay, Richard; Kulik, Liudmila; Ljubanovi?, Danica; Holers, V. Michael

2013-01-01

291

Variability in telavancin cross-reactivity among vancomycin immunoassays.  

PubMed

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

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

2014-12-01

292

Detection of petroleum hydrocarbons in soil and water by immunoassay  

SciTech Connect

A magnetic particle based enzyme immunoassay (EIA) that detects small aromatic hydrocarbons was developed. This EIA can be used to directly test water samples or can be coupled with a simple extraction method to identify the presence of petroleum hydrocarbons in soil. This immunoassay offers several advantages over traditional testing methods (i.e. GC) including speed, cost effectiveness and portability. This assay can be performed on site in less than one hour. The assay`s performance with soil and water samples was evaluated in several studies. In one study conducted on water samples from locations across the US, recoveries of spiked Total BTEX averaged greater than 99% with results ranging from 87% to 119%; one false positive (1.8%) was observed. Soil samples spiked with Total BTEX at concentrations ranging from 0.25 to 10 ppm were extracted, diluted and evaluated in the immunoassay. Recoveries averaged 113% with results ranging from 104% to 120%. In a third study, soil samples collected from various remediation sites were extracted and run in the immunoassay. The assay and recommended extraction procedure agreed well with results obtained by EPA Method 8020 in determining the presence and degree of contamination. Additional study results and data on the cross-reactivity of the assay for various small aromatic hydrocarbons and petroleum fuel mixtures (i.e. gasoline) are also presented.

Selisker, M.Y.; McCaffrey, C.R.; Herzog, D.P.; Itak, J.A. [Ohmicron Corp., Newtown, PA (United States)

1995-12-31

293

Immunoassays DOI: 10.1002/anie.200502862  

E-print Network

Immunoassays DOI: 10.1002/anie.200502862 Label-Free Affinity Assays by Rapid Detection of Immune-based sensing of specific complexes may enable portable or high-throughput immunoassays for diagnostics

Mayer, Michael

294

Field Analytic Technologies: Immunoassay and Enzymatic Assays  

NSDL National Science Digital Library

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

295

Mass spectrometric immunoassay.  

PubMed

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. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. 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 form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus. PMID:15134097

Nelson, R W; Krone, J R; Bieber, A L; Williams, P

1995-04-01

296

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...

297

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

PubMed Central

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p?=?0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies. PMID:24386329

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

2013-01-01

298

Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay.  

PubMed

Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed. PMID:1999653

Kenigsberg, R L; Elliott, P J; Cuello, A C

1991-02-15

299

The importance of IgM positivity in laboratory diagnosis of gestational and congenital syphilis  

PubMed Central

From January 1, 2009 through December 31, 2011, from 33,753 blood samples for syphilis screening, Treponema pallidum infections were confirmed in 241 pregnant women at the Department of Dermatology, Venerology, and Dermatooncology of Semmelweis University Budapest. In this period, four children born to inadequately or untreated women were confirmed to have connatal syphilis. The height of rapid plasma reagin (RPR) titer was measured to determine the stage of the infection and to examine the success of the antilues therapy. The diagnosis of maternal syphilis infection was confirmed with enzyme linked immunosorbent assay (ELISA), T. pallidum particle agglutination (TPPA), and IgG and IgM immunoblots. Maternal IgM immunoblot results identify mothers at risk of delivering babies with connatal syphilis better than the height of maternal RPR titer. The standard serological tests are less useful in newborns because of IgG transfer across the placenta. IgM test which depends on the infant’s response has more specificity in diagnosing connatal syphilis. PMID:24672684

Nemes-Nikodém, É.; Vörös, E.; Pónyai, K.; Párducz, L.; Kárpáti, S.; Rozgonyi, F.; Ostorházi, E.

2012-01-01

300

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

2010-11-01

301

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

SciTech Connect

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

Dautlick, J.X.; Teaney, G.B.; Hudak, R.T.; Melby, J.M. [Strategic Diagnostics Industries Inc., Newark, DE (United States)

1995-12-31

302

Evaluation of immunoassays for the measurement of erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport  

Microsoft Academic Search

The measurement of serum erythropoietin (EPO) has been proposed as one of the indirect biomarkers for the detection of recombinant human EPO misuse in sport. An extended inter-laboratory validation of two commercial immunoassays for EPO measurement is described. A chemiluminescent immunoassay kit (CHEM) and an enzyme-linked immunosorbent assay kit (ELISA) were evaluated.The CHEM assay showed intra-laboratory precision better than 6%

Rosario Abellan; Rosa Ventura; Simona Pichini; Angel Francisco Remacha; Jose Antonio Pascual; Roberta Pacifici; Rita Di Giovannandrea; Piergiorgio Zuccaro; Jordi Segura

2004-01-01

303

IgM, Fc mu Rs, and malarial immune evasion.  

PubMed

IgM is an ancestral Ab 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 Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have 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 mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies. PMID:20410497

Czajkowsky, Daniel M; Salanti, Ali; Ditlev, Sisse B; Shao, Zhifeng; Ghumra, Ashfaq; Rowe, J Alexandra; Pleass, Richard J

2010-05-01

304

The biological effects of IgM hexamer formation.  

PubMed

The inducible B cell lymphoma, CH12, and its in vitro adapted subclone, CH12-LBK, produce immunoglobulins of identical sequence, specificity and isotype, with equivalent affinities for the hapten trimethyl ammonium. However, the hemolytic efficiencies of the antibody secreted by the two cell lines are quite different. Antibody preparations from lipopolysaccharide-stimulated CH12 cells lyse erythrocytes six- to ten times more effectively than antibody preparations of the same concentration from CH12-LBK cells. Both cell lines secrete polymeric IgM, but while CH12-LBK cells secrete predominantly the canonical pentameric IgM, CH12 cells secrete a mixture of pentamers and hexamers. High-efficiency complement-dependent cytolysis is associated with hexameric IgM, which has a specific activity that is approximately twenty times higher than that of the pentameric form. J chain protein is found in the secreted IgM of both cell lines, but is associated only with the pentameric IgM and not with the hexameric form, nor with any intermediate polymers smaller than a pentamer. A deficit in, or the inaccessibility of, J chain protein appears to facilitate hexamer formation. These experiments confirm previously published data showing that J chain is not necessary either for assembly or secretion of polymeric IgM, and suggest instead that J chain may be important in regulating the lytic efficiency of polymeric IgM by controlling the IgM pentamer/hexamer ratio. The experiments further suggest a mechanism, in addition to isotype switching and somatic mutation, by which the biological efficiency of antibodies from a single clone of B cells can be regulated. PMID:2120070

Randall, T D; King, L B; Corley, R B

1990-09-01

305

IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS  

EPA Science Inventory

This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

306

Synthesis of Haptens and Derivation of Monoclonal Antibodies for Immunoassay of the Phenylurea Herbicide Diuron  

Microsoft Academic Search

Diuron and related phenylurea herbicides and their metabolites are important candidates for sensitive and specific immunodetection. This paper describes a scheme for the synthesis of two different types of phenylurea haptens for immunization and use as detecting conjugates in enzyme immunoassays (EIAs). The haptens were used to develop indirect and direct EIAs and to derive a panel of monoclonal antibodies

Alexander E. Karu; Marvin H. Goodrow; Douglas J. Schmidt; Bruce D. Hammock; Michael W. Bigelow

1994-01-01

307

Application and validation of polybrominated diphenyl ethers immunoassay for environmental and food matrices.  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

308

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

Technology Transfer Automated Retrieval System (TEKTRAN)

Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for seru...

309

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

2012-01-01

310

Enhancing immunoassay possibilities using magnetic carriers in biological fluids  

NASA Astrophysics Data System (ADS)

An antibody-based magnetic plate chemifluorescent immunoassay (MPFIA) for effective and rapid detection of bacteria and toxoid from biological fluids was developed. Streptavidin (SA)- magnetic particles and biotinylated antibody as a solid phase immunomagnetic carrier was used for antigen capture. An alkaline phosphatase-antibody conjugate as a secondary capture antibody to the antigen forms a sandwich with the primary antibody. The fluorgenic substrate, AttoPhos reacts with alkaline phosphatase that emits chemifluorescent intensities are proportional to captured antigens. Antigen separation and concentration from biological fluids using immunomagnetic carrier are the key step to reduce media interference for sensitive detection. Results of these efforts may actually enhance the immunoassay possibilities by concentration of specific antigen and reduction of background noise. Magnetic separation and chemifluorescent detection have been achieved by a multiple-well formatted magnetic plate separator and a fluorescent plate detector, respectively. Experiments were performed for virulent Escherichia coli cells, Staphylococcal enterotoxin B toxoid and Bacillus subtilus spore detection in biological fluids. In general, the fluorescent detection can be achieved at the same sensitivities as enzyme-linked immunoassay and the ECL detection is more sensitive than fluorescent assay. However, the unique features of MPFIA and MPECL are that the biological samples can be rapidly processed and detected on the same multiple sample formatted plate within one hour assay time.

Yu, Hao

1997-05-01

311

Pholcodine interference in the immunoassay for opiates in urine.  

PubMed

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

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

1983-01-01

312

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

PubMed Central

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

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

2015-01-01

313

Validation of a microsphere immunoassay for serological leptospirosis diagnosis in human serum by comparison to the current gold standard.  

PubMed

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

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

2015-03-01

314

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

PubMed Central

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

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

2011-01-01

315

Homogeneous fluorescence polarization immunoassay for eicosanoids  

Microsoft Academic Search

Homogeneous fluorescence polarization immunoassay (FPIA) was applied to the development of an immunoassay of prostaglandin E1 (PGE1) selected as a model of eicosanoids. PGE1 was derivatized with fluoresceinthiocarbamyl ethylenediamine and purified by thin layer chromatography (TLC). The purity was checked by high performance liquid chromatography (HPLC). A radioimmunoassay was realized to ensure of the immunocompetition of the tracer. After the

M. Matt; A. Nicolas; M. Donner; J. F. Stoltz

1992-01-01

316

Fluorescence Polarization Immunoassay of Mycotoxins: A Review  

Technology Transfer Automated Retrieval System (TEKTRAN)

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 Kdal), immunoassays for their d...

317

Immunoassay based water quality analysis: A new tool for drinking water supply management  

SciTech Connect

The recent availability of enzyme-linked immunosorbent assay (ELISA) tests for the analysis of organic environmental contaminants provides drinking water utility managers and operators with a new tool for managing treatment operations and monitoring source watersheds. Immunoassay technology permits rapid, inexpensive and accurate in-plant testing of many SDWA regulated organic contaminants at concentrations well below established MCL`s. Analytical testing which would not be practicable due to the high cost or long turnaround time limitations of conventional testing methods is now being performed using immunoassay based analysis. Water quality data generated using immunoassay based methods are being utilized by drinking water utilities as an integral part of source watershed management programs, process operations optimization efforts, pro-active raw and finished water testing programs, and flood and incident response management.

Kostyshyn, C.R. [Ohmicron Environmental Diagnostics, Inc., Newton, PA (United States); Brown, W.; Hervey, E. [City Water Light and Power, Springfield, IL (United States); Hull, C. [Water & Pollution Control Department, Kanasa City, MO (United States)] [and others

1996-11-01

318

BTEX site assessment by D TECH{trademark} immunoassay: A rapid and cost-effective technique  

SciTech Connect

Recent advances in immunoassay, a technique widely accepted as the method of choice for in-vitro diagnostics, have greatly contributed to the detection of environmental contamination. The development of assays for various contaminants, including BTEX, has allowed for an easy and rapid site characterization allows for a more accurate site assessment, largely due to the elimination of the volatility and biodegradation concerns related to BTEX analyses. To demonstrate the performance of the enzyme immunoassay, several field studies were conducted with concurrent analyses by both immunoassay and gas chromatography (GC). In these studies, samples were split into two aliquots in the field. One aliquot was analyzed on-site using the D TECH BTEX kit, the other was sent to a certified laboratory for SW-846 method 8020 GC analysis. The results and subsequent costs of each analysis technique are compared.

Hudak, R.T.; Melby, J.M.; Stave, J.W. [Strategic Diagnostics Inc., Newark, DE (United States)

1994-12-31

319

Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors.  

PubMed

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice. PMID:25552715

Qiu, Qi; Xu, Zhili; Tian, Jie; Moitra, Rituparna; Gunti, Sreenivasulu; Notkins, Abner L; Byrnes, Andrew P

2015-03-15

320

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

321

Preprogrammed, Parallel On-Chip Immunoassay Using System-Level Capillarity Control  

PubMed Central

Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources. PMID:23789820

Kim, Sung-Jin; Paczesny, Sophie; Takayama, Shuichi; Kurabayashi, Katsuo

2014-01-01

322

The development of immunoassays for detection of chemical warfare agents  

SciTech Connect

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

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

1995-06-01

323

Immunoassay for the quantitation of human leukocyte interferon.  

PubMed

A sensitive nonradioactive immunoassay based on monoclonal antibodies was developed. A number of monoclonal hybridomas secreting antibodies against human leukocyte interferon (IFN alpha) were generated using mice immunized with purified lymphoblastoid IFN. Although the binding of antibody to IFN alpha was used as one criterium of selection, all antibodies found can neutralize its antiviral activity. A pair of antibodies binding to different regions of IFN alpha was identified. These were incorporated into sensitive sandwich assays of IFN alpha. A microtiter plate assay - using horse radish peroxidase as marker enzyme - is able to detect IFN alpha at a concentration of 30 IU/ml within 5 h. An overnight tube assay can detect approximately 30 pg IFN alpha 2 or 3 IU per ml solution. The 5-h ELISA (enzyme-linked immunosorbent assay) is well suited for the monitoring of the recovery of rIFN alpha from recombinant organism, during purification and refinement of the protein to a therapeutic drug. PMID:4039175

Berthold, W; Merk, W; Adolf, G R

1985-01-01

324

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

325

Species cross-reactivity of rheumatoid factors and implications for immunoassays.  

PubMed

Rheumatoid factors (RFs) are antibodies recognizing other antibodies usually by binding to the Fc part, while heterophilic antibodies (HAbs) are antibodies reacting with immunoglobulins (Igs) from other species. In particular, RFs have been found to cause false positive results in sandwich immunoassays. In this work, we analyzed RF-positive and RF-negative sera for content of cytokines and for heterophilic reactions by enzyme-linked immunosorbent assay and bead-based sandwich immunoassays. All sera, including those with RFs, contained insignificant amounts of cytokines and chemokines, but RF-positive sera showed large false positive values for several cytokines when analyzed by fluorescent bead-based multiplex immunoassays. This non-specific binding could be minimized by reagents designed to block HAbs, i.e. by selected animal IgGs. Furthermore, sera positive for RFs reacted with several animal IgGs, when these were immobilized on beads or coated on the polystyrene surface in enzyme-linked immunosorbent assays. This reaction could be inhibited by human IgG and by agents designed to inhibit heterophilic reactions (i.e. mixtures of IgGs from different species). In conclusion, RFs and HAbs represent an identical/overlapping set of antibodies, causing false positive reactions in sandwich and other immunoassays. Such assays must be conducted in the presence of appropriate blocking agents, e.g. HBR+, and must be carefully controlled. PMID:25347362

Holm, Bettina E; Sandhu, Noreen; Tronstrøm, Julie; Lydolph, Magnus; Trier, Nicole H; Houen, Gunnar

2015-01-01

326

Polyreactive igm antibodies in the circulation are masked by antigen binding  

Microsoft Academic Search

Human plasma containing IgM showed only minimal, if any, reactivity with a panel of antigens as measured by ELISA. In contrast, affinity-purified IgM showed many times more reactivity with the same panel of antigens. When plasma was added back to the affinity-purified IgM, the reactivity of the IgM with antigens was completely inhibited by undiluted plasma and by as much

George Sigounas; Nick Kolaitis; Esteban Monell-Torrens; Abner Louis Notkins

1994-01-01

327

Enzyme immunoassay using BCG in serodiagnosis of pulmonary tuberculosis.  

PubMed Central

Amounts of Mycobacterium tuberculosis antibodies were determined in sera from patients with either active or inactive tuberculosis and in healthy subjects by an immunoenzymatic assay in which whole BCG cells attached covalently to polystyrene disks were used as antigen. Statistically significant differences (P less than 0.005) were found both between the active and inactive tuberculosis groups and between the active group and healthy controls. No significant differences were found between the inactive group and controls. Since this procedure is efficient (91%) and can be used in areas which lack laboratory equipment, it appears promising for individual serodiagnosis and for epidemiological surveys. PMID:3098836

Garcia-Carreño, F. L.; Carvajal, R. E.; Hernandez, R.

1986-01-01

328

Enzyme immunoassay for myelin basic protein in cerebrospinal fluid  

Microsoft Academic Search

Myelin basic proteins (MBPs) are a set of proteins making up about 30% of the protein content of the central nervous system myelin. Four human isoforms have been identified [11]. The most abundant is a highly conserved 18.5 kDa polypeptide. For this species, the amino acid sequence homologies between human and monkey or human and chick are 98.2% and 71.1%,

Farid Najeme; Jean Julien; Sabine Herblot; Vincent Dousset; Bruno Brochet; Jacques Bonnet

1997-01-01

329

[Performance of the VIDAS automated immunoassay for the determination of Epstein-Barr virus serological status].  

PubMed

Enzyme linked fluorescent assays (VIDAS EBV VCA IgM, VIDAS EBV VCA/EA IgG and VIDAS EBV EBNA IgG (Biomérieux, France) were evaluated to determine markers for infection of Epstein Barr virus, as well as to establish antibody profiles, compared with immunofluorescence assays as reference. The assays evaluated showed good values for sensitivity, specificity and agreement, making them useful for their application in clinical laboratories. PMID:22854635

De Ory, Fernando; Guisasola, María Eulalia; Sanz, Juan Carlos; García-Bermejo, Isabel

2012-12-01

330

Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.  

PubMed

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

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

2015-01-01

331

Serological Responses to Experimental Norwalk Virus Infection Measured Using a Quantitative Duplex Time-Resolved Fluorescence Immunoassay ?  

PubMed Central

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

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

2011-01-01

332

Evaluation of the Vitros Syphilis TPA Chemiluminescence Immunoassay as a First-Line Method for Reverse Syphilis Screening.  

PubMed

We report here the results of the diagnostic performances of Vitros Syphilis TPA (a chemiluminescence treponemal assay) compared with those of two treponemal enzyme immunoassays and of traditional versus reverse syphilis algorithms. Ease of use, automation, and high throughput make the Vitros Syphilis TPA assay a good choice for syphilis screening in high-volume laboratories. PMID:25609729

González, Victoria; Fernández, Gema; Dopico, Eva; Margall, Nuria; Esperalba, Juliana; Muñoz, Carme; Castro, Elisabeth; Sulleiro, Elena; Matas, Lurdes

2015-04-01

333

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

EPA Science Inventory

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

334

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

335

IGM Heating and AGN activity in Fossil Galaxy Groups  

NASA Astrophysics Data System (ADS)

Fossil galaxy groups are energetically and morphologically ideal environments to study the intergalactic medium (IGM) heating, because their inter-galactic gas is undisturbed due to the lack of recent group scale mergers. We study the role of active galactic nuclei (AGN) in heating the IGM in a sample of five fossil galaxy groups by employing properties at 610 MHz and 1.4 GHz. We find that two of the dominant galaxies in fossil groups, ESO 3060170 and RX J1416.4+2315, are associated with the radio lobes. We evaluate the PdV work of the radio lobes and their corresponding heating power and compare to the X-ray emission loss within cooling radius. Our results show that the power due to mechanical heating is not sufficiently high to suppress the cooling.

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

2014-07-01

336

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

PubMed Central

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

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

2014-01-01

337

ENZYME: Enzyme Nomenclature Database  

NSDL National Science Digital Library

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

338

Tube-Immunoassay for Rapid Detection of Carbaryl Residues in Agricultural Products  

Microsoft Academic Search

An antibody-based rapid, quantitative, and qualitative tube enzyme-linked immunosorbent assay (tube-ELISA) was developed and used to determine carbaryl (1-naphthyl methylcarbamate) residues in agricultural products (apple, Chinese cabbage, rice, and barley). The tube-ELISA is a competitive immunoassay in which the antibody is coated in the polystyrene tube, with a dynamic range between 0.7 and 46.3 ?g kg. Carbaryl was extracted from

SHUO WANG; CHUNDI YU; YAN ZHANG; JUNPING WANG; ZHENJUAN DUAN; JUANKUN ZHANG

2006-01-01

339

A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies  

PubMed Central

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 enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra- and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations. PMID:10681316

Machard, Sophie; Theodoro, Frederic; Benech, Henri; Grognet, Jean-Marc; Ezan, Eric

2000-01-01

340

Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.  

PubMed

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner. PMID:21086008

Shoma, S; Verkaik, N J; de Vogel, C P; Hermans, P W M; van Selm, S; Mitchell, T J; van Roosmalen, M; Hossain, S; Rahman, M; Endtz, H Ph; van Wamel, W J B; van Belkum, A

2011-04-01

341

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

PubMed Central

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

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

2012-01-01

342

Detection of IgM and IgG anti-Toxoplasma antibodies in renal transplant recipients using ELFA, ELISA and ISAGA methods: comparison of pre- and post-transplantation status  

PubMed Central

In the transplant recipient patients receive immunosuppressive therapy, the possibility of reactivation of the old infection or acquisition of infection from a donor’s tissue increases. In this study, IgM and IgG anti-Toxoplasma immunoglobulins seroconversion in renal transplant recipients (RTRs) have been evaluated before and after transplantation. This is a prospective cohort study on a total of 102 RTRs. Two serum samples were obtained from each patient. The first was taken before administration of any immunosuppressive drugs such as corticosteroids and the second was taken 3 months after transplantation. The IgM and IgG anti-Toxoplasma antibodies were assayed by enzyme-linked flourescence assay (ELFA) and enzyme-linked immunosorbent assay (ELISA) techniques. IgM/immunosorbent agglutination assay (ISAGA) method has also been used. All RTRs were tested for toxoplasmosis before and after transplantation. ELFA identified 65 (63.7%) pre-transplantation samples as IgG+ and did not detect any positive IgM samples. However, IgM was detected in three (2.9%) post-transplantation samples by this method. Forty-nine (48%) pre-transplantation samples were reported IgG+ by ELISA and no IgM positive sample was identified by this method. ELISA has detected two (1.9%) IgM-positive reactions in post-transplantation samples. By IgM/ISAGA method, we have detected no IgM positive reactions in pre-transplantation samples, whereas 3 months later (second sampling) IgM antibody was detected in 3 (2.9%) cases. Secondary toxoplasmosis infection was observed in 30 cases per 1000 RTRs, which indicates that screening for toxoplasmosis infection should be performed in developed countries for these patients. On the other hand, as the risk of re-active toxoplasmosis infection exists in developing nations, they should consider the necessary preventive measures to control this condition. PMID:21929878

GHARAVI, M J; JALALI, S; KHADEMVATAN, S; HEYDARI, S

2011-01-01

343

Detection of IgM and IgG anti-Toxoplasma antibodies in renal transplant recipients using ELFA, ELISA and ISAGA methods: comparison of pre- and post-transplantation status.  

PubMed

In the transplant recipient patients receive immunosuppressive therapy, the possibility of reactivation of the old infection or acquisition of infection from a donor's tissue increases. In this study, IgM and IgG anti-Toxoplasma immunoglobulins seroconversion in renal transplant recipients (RTRs) have been evaluated before and after transplantation. This is a prospective cohort study on a total of 102 RTRs. Two serum samples were obtained from each patient. The first was taken before administration of any immunosuppressive drugs such as corticosteroids and the second was taken 3 months after transplantation. The IgM and IgG anti-Toxoplasma antibodies were assayed by enzyme-linked flourescence assay (ELFA) and enzyme-linked immunosorbent assay (ELISA) techniques. IgM/immunosorbent agglutination assay (ISAGA) method has also been used. All RTRs were tested for toxoplasmosis before and after transplantation. ELFA identified 65 (63·7%) pre-transplantation samples as IgG+ and did not detect any positive IgM samples. However, IgM was detected in three (2·9%) post-transplantation samples by this method. Forty-nine (48%) pre-transplantation samples were reported IgG+ by ELISA and no IgM positive sample was identified by this method. ELISA has detected two (1·9%) IgM-positive reactions in post-transplantation samples. By IgM/ISAGA method, we have detected no IgM positive reactions in pre-transplantation samples, whereas 3 months later (second sampling) IgM antibody was detected in 3 (2·9%) cases. Secondary toxoplasmosis infection was observed in 30 cases per 1000 RTRs, which indicates that screening for toxoplasmosis infection should be performed in developed countries for these patients. On the other hand, as the risk of re-active toxoplasmosis infection exists in developing nations, they should consider the necessary preventive measures to control this condition. PMID:21929878

Gharavi, M J; Jalali, S; Khademvatan, S; Heydari, S

2011-07-01

344

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

2013-01-01

345

Development of an Ultrasensitive Immunoassay for Detecting Tartrazine  

PubMed Central

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

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

2013-01-01

346

The development of immunoassays for detection of chemical warfare agents  

SciTech Connect

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

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

1996-10-01

347

Electrothermal stirring for heterogeneous immunoassays Marin Sigurdson,a  

E-print Network

Electrothermal stirring for heterogeneous immunoassays Marin Sigurdson,a Dazhi Wangb and Carl D-sensors, for example, immunoassays, in which a ligand immobilized on a microchannel wall specifically binds analyte of these heterogeneous immunoassays may be improved by using AC electrokinetically-driven microscale fluid motion

Meinhart, Carl

348

Ultrashort Separation Length Homogeneous Electrophoretic Immunoassays Using On-Chip  

E-print Network

Ultrashort Separation Length Homogeneous Electrophoretic Immunoassays Using On-Chip Discontinuous-color homogeneous electrophoretic immunoassay for concur- rent quantitation of C reactive protein (CRP) and tumor for near-patient environments. Homogeneous electrophoretic immunoassays are well-suited for and often

Herr, Amy E.

349

Ultra-sensitive immunoassay using VCSEL detection system  

E-print Network

Ultra-sensitive immunoassay using VCSEL detection system C.F.R. Mateus, M.C.Y. Huang, C.J. Chang. In this Letter, we report the use of this system in immunoassay with a record high sensitivity to antigen be largely extended (32 nm) by using a MEMS tunable VCSEL [4]. Mouse IgG capture immunoassay: In this Letter

Cunningham, Brian

350

PROBLEM 6: MASS TRANSPORT AND SURFACE REACTION OF IMMUNOASSAY  

E-print Network

PROBLEM 6: MASS TRANSPORT AND SURFACE REACTION OF IMMUNOASSAY Jim Buchanan, Henrik Kalisch applications, the solid phase surface reactions and immunoassay technologies are widely used to determine of the Y. Antigen attached with a label is used in a variety of immunoassay techniques. The labeled antigen

Kalisch, Henrik

351

Application of Photonic Crystal Enhanced Fluorescence to a Cytokine Immunoassay  

E-print Network

Application of Photonic Crystal Enhanced Fluorescence to a Cytokine Immunoassay Patrick C. Mathias on the glass slidesa decrease from 18 to 6 pg/mL. The increased performance of the immunoassay allows for more detection has been an adaptation of the DNA microarray format to immunoassays. Fluorescence-based protein

Cunningham, Brian

352

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria  

Microsoft Academic Search

A tandem technique for the detection of very low levels E. coli within about 2h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli ?-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay

Ramadan A. Abuknesha; Fatima Darwish

2005-01-01

353

COMPARATIVE ABSORPTION OF COLOSTRAL IgG1 AND IgM IN THE NEWBORN CALF  

E-print Network

COMPARATIVE ABSORPTION OF COLOSTRAL IgG1 AND IgM IN THE NEWBORN CALF EFFECTS OF THYROXINE, CORTISOL Nutritionnelles, l.N.R.A., Centre de Theix, 63110 Beaumont France Résumé ABSORPTION DES IgGl ET IgM COLOSTRALES conditions. Les résultats suivants ont été obtenus : - la capacité d'absorption des IgGl et des IgM varie

Boyer, Edmond

354

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

355

Heavy element enrichment in the IGM at high redshift  

E-print Network

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

S. Savaglio

1997-09-16

356

Immunoassays for trifloxystrobin analysis. Part II. Assay development and application to residue determination in food.  

PubMed

Immunochemical assays constitute complementary analytical methods for small organic molecule determination. We herein describe the characterisation and optimisation of two competitive enzyme-linked immunosorbent assays in different formats using monoclonal antibodies to the Quinone outside inhibitor (QoI) fungicide trifloxystrobin. Antibody selectivity was evaluated using a variety of agrochemicals and the main trifloxystrobin metabolite. Acceptable tolerance of the immunoassay to methanol, ethanol, and acetonitrile was observed in all cases, whereas a dissimilar influence of buffer pH and ionic strength was found. Moreover, the influence of Tween 20 over the analytical parameters was studied. The limits of detection of the optimised assays were below 0.1 ?g L(-1). Excellent recoveries, even at 10 ?g kg(-1), were obtained when strawberry, tomato, and cucumber samples spiked with trifloxystrobin were analysed. Finally, statistical agreement was found between immunoassay and reference chromatographic results using blind-spiked and in-field treated samples. PMID:24874355

Mercader, Josep V; López-Moreno, Rosario; Esteve-Turrillas, Francesc A; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

2014-11-01

357

Fluorescence Polarization Immunoassay for Insulin Preparations  

Microsoft Academic Search

A fluorescence polarization immunoassay for insulin preparations such as pharmaceutical preparation and solutions after chromatography is described. The assay is rapid, without separation of antibody-bound and free ligands.The minimal amount of insulin detected was 0.6 mU\\/ tube and the measurable range was from 40 to 600 mU\\/ml of solution.

Y. Yamaguchi; C. Hayashi; K. Miyai

1982-01-01

358

Analysis of Sulfamethazine by Fluorescence Polarization Immunoassay  

Microsoft Academic Search

A fluorescence polarization immunoassay (FPIA) was developed based on a polyclonal antibody for the determination and the qualitative screening analysis of sulfamethazine (SMZ). To optimize the FPIA procedures, three fluorescein-labeled SMZ and sulfamerazine (SMR) were synthesized, and the influence of their structures on the characteristics of FPIA was studied. The optimized FPIA for SMZ had a dynamic range from 5

Zhan-Hui Wang; Su-Xia Zhang; Jian-Zhong Shen; A. Eremin Sergei

2007-01-01

359

Monitoring human exposure to pesticides using immunoassay  

E-print Network

Monitoring human exposure to pesticides using immunoassay Marja E. Koivunen1,2 , Shirley J. Gee1, specific and sensitive tools for human exposure studies involving numerous samples in complex matrices (4 compound requires extensive knowledge about its biotransformation and metabolism in humans. Studies

Hammock, Bruce D.

360

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...

361

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone.  

PubMed

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP. PMID:2175328

Bost, K L; Clarke, B L; Xu, J C; Kiyono, H; McGhee, J R; Pascual, D

1990-12-15

362

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

PubMed

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

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

2009-09-25

363

Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker  

PubMed Central

Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the working range was 0.1–11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%–6.5% (ICA-PLAP assay) and 9.0%–9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes. PMID:17489017

Stinghen, Sérvio T.; Moura, Juliana F.; Zancanella, Patrícia; Rodrigues, Giovanna A.; Pianovski, Mara A.; Lalli, Enzo; Arnold, Dodie L.; Minozzo, João C.; Callefe, Luis G.; Ribeiro, Raul C.; Figueiredo, Bonald C.

2006-01-01

364

Recovering the topology of the IGM at z~2  

E-print Network

We investigate how well the 3D density field of neutral hydrogen in the Intergalactic Medium (IGM) can be reconstructed using the Lyman-alpha absorptions observed along lines of sight to quasars separated by arcmin distances in projection on the sky. We use cosmological hydrodynamical simulations to compare the topologies of different fields: dark matter, gas and neutral hydrogen optical depth and to investigate how well the topology of the IGM can be recovered from the Wiener interpolation method implemented by Pichon et al. (2001). The global statistical and topological properties of the recovered field are analyzed quantitatively through the power-spectrum, the probability distribution function (PDF), the Euler characteristics, its associated critical point counts and the filling factor of underdense regions. The local geometrical properties of the field are analysed using the local skeleton by defining the concept of inter-skeleton distance. At scales larger than ~1.4 , where is the mean separation between lines of sight, the reconstruction accurately recovers the topological features of the large scale density distribution of the gas, in particular the filamentary structures. At scales larger than the intrinsic smoothing length of the inversion procedure, the power spectrum of the recovered HI density field matches well that of the original one and the low order moments of the PDF are well recovered as well as the shape of the Euler characteristic. The integral errors on the PDF and the critical point counts are indeed small, less than 20% for ~2.5 arcmin. The small deviations between the reconstruction and the exact solution mainly reflect departures from the log-normal behaviour that are ascribed to highly non-linear objects in overdense regions.

S. Caucci; S. Colombi; C. Pichon; E. Rollinde; P. Petitjean; T. Sousbie

2008-01-28

365

IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients.  

PubMed

Melanoma cells express ganglioside antigens GM3, GD3, GM2, and GD2 on their surface. This study examined whether immunization with a melanoma cell vaccine induced anti-ganglioside antibody responses in melanoma patients and whether these responses were correlated with survival. Sixty-six patients who had received melanoma cell vaccine immunotherapy after surgical removal of regional metastatic melanoma were identified. Cryopreserved serum samples from these patients were used in an enzyme-linked immunsorbent assay to determine the IgM antibody levels to GM2, GD2, GM3, and GD3 prior to melanoma cell vaccine treatment and 4 wk after the first melanoma cell vaccine immunization. All antibody levels significantly increased by week 4 (p < 0.001 for all four antibodies) and all increases were significantly associated with survival (anti-GD2, p < 0.001; anti-GM2, p = 0.001; anti-GD3, p < 0.001; anti-GM3, p < 0.001). Anti-tumor activity of these antibodies was proved using five representative antibody-positive sera in a complement-dependent cytotoxicity assay with cultured melanoma cell lines. These studies suggest that GM2, GD2, GM2, and GD3 expressed by melanoma cells can induce specific IgM antibodies and that high levels of these antibodies might have a beneficial impact on survival. PMID:9989797

Takahashi, T; Johnson, T D; Nishinaka, Y; Morton, D L; Irie, R F

1999-02-01

366

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

E-print Network

1 The Insertion Green Monster (iGM) Method for Expression of Multiple Exogenous Genes in Yeast;2 Running title: The Insertion Green Monster Method Key words: synthetic biology, multi-gene insertions Green Monster' (iGM) set of expression vectors that enable precise insertion of many heterologous genes

Roth, Frederick

367

The importance of natural IgM: scavenger, protector and regulator  

Microsoft Academic Search

The existence of IgM has been known for more than a century, but its importance in immunity and autoimmunity continues to emerge. Studies of mice deficient in secreted IgM have provided unexpected insights into its role in several diverse processes, from B cell survival to atherosclerosis, as well as in autoimmunity and protection against infection. Among the various distinct properties

Clare A. Notley; Michael R. Ehrenstein

2010-01-01

368

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. ...

369

Laboratory diagnosis of syphilis with automated immunoassays.  

PubMed

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

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

2009-01-01

370

Alkaline phosphatase as a label for immunoassay using amperometric detection with a variety of substrates and an optimal buffer system  

Microsoft Academic Search

Novel substrates for use in an amperometric 3-electrode system are described for the determination of alkaline phosphatase (EC.3.1.3.1), the enzyme label most commonly used in electrochemical immunoassays. Previous problems encountered with a variety of substrates have led to passivation of the working electrode at low product concentrations. Our group has synthesised a number of novel substrates in an attempt to

M. P. Kreuzer; C. K. O'Sullivan; G. G. Guilbault

1999-01-01

371

Development of a magnetic particle immunoassay for polybrominated diphenyl ethers and application to environmental and food matrices  

Microsoft Academic Search

A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about 1h after sample cleanup. The assay has a limit of detection (LOD) below 0.1ppb towards the following brominated diphenyl ether (BDE) congeners: BDE-47, BDE-99,

Weilin L. Shelver; Carmen D. Parrotta; Richard Slawecki; Qing X. Li; Michael G. Ikonomou; Damià Barcelo; Silvia Lacorte; Fernando M. Rubio

2008-01-01

372

Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms.  

PubMed

Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2? or Ab2?). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin. PMID:24526340

Maragos, C M

2014-05-01

373

Hybridization chain reaction-based fluorescence immunoassay using DNA intercalating dye for signal readout.  

PubMed

A novel format of fluorescence immunosorbent assay based on the hybridization chain reaction (HCR) using a DNA intercalating dye for signal readout was constructed for the sensitive detection of targets, both in competitive and sandwich modes. In this platform, the capture and recognition processes are based on immunoreactions and the signal amplification depends on the enzyme-free, isothermal HCR-induced labelling event. After a competitive or a sandwich immunoreaction, a biotinylated capture DNA was bound to a biotinylated signal antibody through avidin, and triggered the HCR by two specific hairpins into a nicked double helix. Gene Finder (GF), a fluorescent probe for double-strand DNA, was intercalated in situ into the amplified chain to produce the fluorescence signal. The limit of detection (LOD) for rabbit IgG in competitive mode by HCR/GF immunoassay was improved at least 100-fold compared with the traditional fluorescence immunoassay using the fluorescein isothiocyanate-labelled-streptavidin or fluorescein isothiocyanate-labelled second antibody as the signal readout. The proposed fluorescence immunoassay was also demonstrated by using ?-fetoprotein as the model target in sandwich mode, and showed a wide linear range from 28 ng mL(-1) to 20 ?g mL(-1) with a LOD of 6.0 ng mL(-1). This method also showed satisfactory analysis in spiked human serum, which suggested that it might have great potential for versatile applications in life science and point-of-care diagnostics. PMID:24828400

Deng, Yan; Nie, Ji; Zhang, Xiao-hui; Zhao, Ming-Zhe; Zhou, Ying-Lin; Zhang, Xin-Xiang

2014-07-01

374

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

375

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

PubMed

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

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

2009-03-01

376

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

PubMed Central

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

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

2014-01-01

377

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

NASA Astrophysics Data System (ADS)

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

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

1997-02-01

378

B-1 cells in the bone marrow are a significant source of natural IgM  

PubMed Central

Summary Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies had identified bone marrow and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM secreting cells in spleen, and for the first time in the bone marrow as IgM+ IgDlo/?CD19hi CD43+ CD5+/? B-1 cells. The newly identified population of bone marrow B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and bone marrow B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Together the study identifies populations of non-terminally differentiated B-1 cells in spleen and bone marrow as the most significant producers of natural IgM. PMID:22009734

Choi, Youn Soo; Dieter, Jacquelyn A.; Rothaeusler, Kristina; Luo, Zheng; Baumgarth, Nicole

2012-01-01

379

The new ParaDIgm: IgM from bench to clinic  

PubMed Central

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

Hanala, Sherif

2012-01-01

380

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

E-print Network

Hapten Synthesis and Antibody Development for Polychlorinated Dibenzo-p-dioxin Immunoassays James R. Keywords: TCDD; dioxin; immunoassay; polyclonal antibodies; hapten synthesis; polychlorinated hydrocarbons

Hammock, Bruce D.

381

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

2008-11-01

382

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

PubMed

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

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

2013-08-15

383

Expression and glycoengineering of functionally active heteromultimeric IgM in plants  

PubMed Central

IgM antibodies are an important player of the human’s innate defense mechanisms and increasingly have gained interest as therapeutics. Although the expression of IgM antibodies in mammalian cell culture is established, this approach remains costly and alternative methods have not been developed yet. Plants have a proven record for the production of therapeutically relevant recombinant proteins. However, whether they are able to express proteins like IgM antibodies, which range among the most complex human proteins, remains unknown so far. Here we report the in planta generation of the functionally active monoclonal antitumor IgM PAT-SM6 (SM6). SM6 efficiently accumulates in plant leaves and assembles correctly into heterooligomers (pentamers and hexamers). Detailed glycosylation analysis exhibited complex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line–produced SM6. Moreover, extensive in planta glycoengineering allowed the generation of SM6 decorated with sialylated human-type oligosaccharides, comparable to plasma-derived IgM. A glycosylated model of pentameric IgM exhibits different accessibility of the glycosylation sites, explaining site-specific glycosylation. Biochemical and biophysical properties and importantly biological activities of plant-derived SM6 glycoforms are comparable to the human-cell–derived counterparts. The in planta generation of one of the most complex human proteins opens new pathways toward the production of difficult-to-express proteins for pharmaceutical applications. Moreover, the generation of IgMs with a controlled glycosylation pattern allows the study of the so far unknown contribution of sugar moieties to the function of IgMs. PMID:24706782

Loos, Andreas; Gruber, Clemens; Altmann, Friedrich; Mehofer, Ulrich; Hensel, Frank; Grandits, Melanie; Oostenbrink, Chris; Stadlmayr, Gerhard; Furtmüller, Paul G.; Steinkellner, Herta

2014-01-01

384

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

PubMed Central

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

Abós, Beatriz; Castro, Rosario; Pignatelli, Jaime; Luque, Alfonso; González, Lucia; Tafalla, Carolina

2013-01-01

385

Interaction between the IGM and a dwarf galaxy  

NASA Astrophysics Data System (ADS)

Dwarf Galaxies are the most common objects in the Universe and are believed to contain large amounts of dark matter. There are mainly three morphologic types of dwarf galaxies: dwarf ellipticals, dwarf spheroidals and dwarf irregulars. Dwarf irregular galaxies are particularly interesting in dwarf galaxy evolution, since dwarf spheroidal predecessors could have been very similar to them. Therefore, a mechanism linked to gas-loss in dwarf irregulars should be observed, i.e. ram pressure stripping. In this paper, we study the interaction between the ISM of a dwarf galaxy and a flowing IGM. We derive the weak-shock, plasmon solution corresponding to the balance between the post-bow shock pressure and the pressure of the stratified ISM (which we assume follows the fixed stratification of a gravitationally dominant dark matter halo). We compare our model with previously published numerical simulations and with the observed shape of the HI cloud around the Ho II and Pegasus dwarf irregular galaxies. We show that such a comparison provides a straightforward way for estimating the Mach number of the impinging flow.

Lora, V.; Raga, A. C.; Grebel, E. K.

2015-04-01

386

IgM nephropathy; can we still ignore it  

PubMed Central

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

Vanikar, Aruna

2013-01-01

387

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

PubMed Central

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

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

2015-01-01

388

Quantitative analysis of plasma interleiukin-6 by immunoassay on microchip  

NASA Astrophysics Data System (ADS)

Sandwich enzyme-linked immunoassay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. We employed the piezoelectric inkjet printing for deposition and fixation of 1st antibody on the microchannnel surface (300 ?m width and 100 ?m depth). Model analyte was interleukin-6 (IL-6) which was one of the inflammatory cytokine. After blocking the microchannel, antigen, biotin-labeled 2nd antibody, and avidin-labeled peroxidase were infused into the microchannel and incubated for 20 min, 10 min, and 5 min, respectively. This assay could detect 2 pg/ml and quantitatively measure the range of 0-32 pg/ml. Liner regression analysis of plasma IL-6 concentration obtained by microchip and conventional methods exhibited a significant relationship (R2 = 0.9964). This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. This assay enables us to determine plasma IL-6 with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

Abe, K.; Hashimoto, Y.; Yatsushiro, S.; Yamamura, S.; Tanaka, M.; Ooie, T.; Baba, Y.; Kataoka, M.

2012-03-01

389

Sensitive Giant Magnetoresistive-based Immunoassay for Multiplex Mycotoxin Detection  

PubMed Central

Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 × 90 µm2, arranged in an 8×8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B1, zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved. PMID:20047828

Mak, Andy C.; Osterfeld, Sebastian J.; Yu, Heng; Wang, Shan X.; Davis, Ronald W.; Jejelowo, Olufisayo A.; Pourmand, Nader

2010-01-01

390

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay.  

PubMed

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids. PMID:3674415

Lee, S R; Liberti, P A

1987-10-01

391

Enzyme Reactions  

NSDL National Science Digital Library

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

Minerva Deland School

2011-10-03

392

Determination of the herbicide acetochlor by fluorescence polarization immunoassay  

Microsoft Academic Search

A fluorescence polarization immunoassay procedure was developed for determining the herbicide acetochlor from the group of chloroacetanilides. Conjugates of fluorescent labeled acetochlor derivatives (tracers) with glycylaminofluorescein and ethylenediaminofluorescein were synthesized. The effect of the tracer structure on the analytical characteristics of the determination and on antigen-antibody binding constants was studied. The developed immunoassay procedures are characterized by a detection limit

M. A. Deryabina; Yu. N. Yakovleva; V. A. Popova; S. A. Eremin

2005-01-01

393

Fluorescence Polarization Immunoassay: Detection of Antibody to Brucella abortus  

Microsoft Academic Search

Fluorescence polarization immunoassay (FPA) is a homogeneous immunoassay useful for rapid and accurate detection of antibody or antigen. The principle of the assay is that a fluorescent dye (attached to an antigen or an antibody fragment) can be excited by plane-polarized light at the appropriate wavelength. As a rule, a small molecule rotates faster when in solution than a larger

Klaus Nielsen; Min Lin; David Gall; Michael Jolley

2000-01-01

394

Immunoassay detection without washing by using AC magnetic susceptibility  

Microsoft Academic Search

Our aim in this study was to demonstrate a stable immunoassay using the decrease of AC magnetic susceptibility without washing to separate bound and unbound magnetic markers. To achieve low noise in the immunoassay system, we examined the arrangement of the MR sensors, the calibration of the distance between the MR sensor and the sample, and the magnetic noise generated

Ryuzo Kawabata; Takako Mizoguchi; Akira Tsukamoto; Tomoko Yoshimura; Akihiko Kandori; Keiji Enpuku

2010-01-01

395

Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective  

Microsoft Academic Search

Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important

J. W. A. Findlay; W. C. Smith; J. W. Lee; G. D. Nordblom; I. Das; B. S. DeSilva; M. N. Khan; R. R. Bowsher

2000-01-01

396

Performance of Indirect Immunoglobulin M (IgM) Serology Tests and IgM Capture Assays for Laboratory Diagnosis of Measles  

PubMed Central

As progress is made toward elimination of measles, the laboratory confirmation of measles becomes increasingly important. However, both false-positive and false-negative results can occur with the routinely used indirect measles immunoglobulin M (IgM) serology tests. The measles IgM capture assay is considered to be more specific, and therefore, its use is indicated for confirmatory testing, but its relative performance has not been fully assessed. Four commercial indirect measles IgM serology test kits (the Behring, Clark, Gull, and PanBio assays) and a commercial IgM capture assay (the Light Diagnostics assay) were evaluated for their abilities to detect measles virus-specific IgM antibody with a total of 308 serum samples from patients involved in a measles outbreak and with confirmed cases of measles and 454 samples from subjects without measles. The Centers for Disease Control and Prevention (CDC) IgM capture assay was also used in a part of the evaluation. Among the indirect assays, the overall sensitivities ranged from 82.8% (Clark assay) to 88.6% (Behring assay) and specificity ranged from 86.6% (PanBio assay) to 99.6% (Gull assay). These rates were 92.2 and 86.6%, respectively, for the Light Diagnostics capture assay and 87.0 and 94.8%, respectively, for the CDC capture assay. While the Light Diagnostics capture assay had the best detection rate (80%) with the acute-phase samples compared with those for the rest of the tests (CDC capture assay, 77%; Behring assay, 70%; Gull assay, 69%; PanBio assay, 58%; and Clark assay, 57%), all tests showed a significantly improved sensitivity in the range of 92% (Clark and PanBio assays) to 97% (Light Diagnostics and CDC capture assays) with the convalescent-phase samples, as expected. The best seropositivity rates (in the range of 92 to 100%) were observed with samples collected 6 to 14 days after the onset of symptoms. The Gull assay showed the highest positive predictive value (99.6%), followed by the Behring assay (97.8%) and the CDC capture assay (96.1%). Overall, the Gull and Behring assays were found to be as good as or better than the capture assays. In conclusion, laboratory diagnosis of measles based on IgM serology varies depending on the timing of specimen collection and the test used, and the case for the use of the IgM capture assay as the confirmatory test appears to be uncertain. PMID:10618071

Ratnam, Samuel; Tipples, Graham; Head, Carol; Fauvel, Micheline; Fearon, Margaret; Ward, Brian J.

2000-01-01

397

Combination assays for IgG class and IgM class anti-pyruvate dehydrogenase complex(PDC)-E2 by ELISA using recombinant autoantigen to diagnose primary biliary cirrhosis  

Microsoft Academic Search

IgG and IgM class anti-pyruvate dehydrogenase complex (PDC)-E2 were studied in sera from anti-M2-positive 84 patients with PBC by enzyme-linked immunosorbent assay (ELISA) to assess the usefulness of combination assays. We used recombinant antigen coding the autoantigenic epitopes of PDC-E2 comprising the lipoic acid binding sites. Antigen specificity of this ELISA was confirmed by inhibition test with pre-incubation of recombinant

Hiroshi Miyakawa; Naomi Kawaguchi; Kentaro Kikuchi; Hirotoshi Fujikawa; Eriko Kitazawa; Masanao Matsushita; Kazuhiro Abe; Makoto Kako

1998-01-01

398

Vesiculovirus Neutralization by Natural IgM and Complement  

PubMed Central

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

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

2014-01-01

399

Interpretation of total petroleum hydrocarbon results with immunoassay test kits  

SciTech Connect

Petroleum derived fuels are complex mixtures of organic compounds, predominantly hydrocarbons, with varying compositions depending on source of the crude oil and its refining process. Results from current analytical methods for petroleum hydrocarbons may be generically referred to as Total Petroleum Hydrocarbons or TPH. Various immunoassay kits for petroleum products are now commercially available. Questions often arise when individuals new to immunoassay technology begin application of kits to these complex mixtures. Many users wish to compare results from the immunoassay screening test to a TPH laboratory results. Studies were conducted to demonstrate the nature of the relationship of common TPH methods with immunoassay methods. The immunoassays studied were developed for determination of volatile aromatic mixtures and for determination of polynuclear aromatic hydrocarbons.

Jourdan, S.; Scutellaro, A.; Hayes, M.; Herzog, D.P. [Ohmicron Environmental Diagnostics, Newtown, PA (United States)

1995-12-31

400

Splenectomy associated changes in IgM memory B cells in an adult spleen registry cohort.  

PubMed

Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited to a spleen registry (n = 591). A subset of 209 adult asplenic or hyposplenic subjects, and normal controls (n = 140) were tested for IgM memory B cells. We also determined a) changes in IgM memory B cells with time after splenectomy using the cross-sectional data from patients on the registry and b) the kinetics of changes in haematological markers associated with splenectomy(n = 45). Total B cells in splenectomy patients did not differ from controls, but memory B cells, IgM memory B cells and switched B cells were significantly (p<0.001) reduced. The reduction was similar for different indications for splenectomy. Changes of asplenia in routine blood films including presence of Howell-Jolly bodies (HJB), occurred early (median 25 days) and splenectomy associated thrombocytosis and lymphocytosis peaked by 50 days. There was a more gradual decrease in IgM memory B cells reaching a stable level within 6 months after splenectomy. IgM memory B cells as proportion of B cells was the best discriminator between splenectomized patients and normal controls and at the optimal cut-off of 4.53, showed a true positive rate of 95% and false positive rate of 20%. In a survey of 152 registry patients stratified by IgM memory B cells around this cut-off there was no association with minor infections and no registry patients experienced OPSI during the study. Despite significant changes after splenectomy, conventional measures of IgM memory cells have limited clinical utility in this population. PMID:21829713

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

2011-01-01

401

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

PubMed Central

Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited to a spleen registry (n?=?591). A subset of 209 adult asplenic or hyposplenic subjects, and normal controls (n?=?140) were tested for IgM memory B cells. We also determined a) changes in IgM memory B cells with time after splenectomy using the cross-sectional data from patients on the registry and b) the kinetics of changes in haematological markers associated with splenectomy(n?=?45). Total B cells in splenectomy patients did not differ from controls, but memory B cells, IgM memory B cells and switched B cells were significantly (p<0.001) reduced. The reduction was similar for different indications for splenectomy. Changes of asplenia in routine blood films including presence of Howell-Jolly bodies (HJB), occurred early (median 25 days) and splenectomy associated thrombocytosis and lymphocytosis peaked by 50 days. There was a more gradual decrease in IgM memory B cells reaching a stable level within 6 months after splenectomy. IgM memory B cells as proportion of B cells was the best discriminator between splenectomized patients and normal controls and at the optimal cut-off of 4.53, showed a true positive rate of 95% and false positive rate of 20%. In a survey of 152 registry patients stratified by IgM memory B cells around this cut-off there was no association with minor infections and no registry patients experienced OPSI during the study. Despite significant changes after splenectomy, conventional measures of IgM memory cells have limited clinical utility in this population. PMID:21829713

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

2011-01-01

402

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients  

Microsoft Academic Search

Abstract. Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross- linking) and low-mutated IgM antibodies (less immunogenic),are believed to be the most effective weapons,against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also

Stephanie Br Andlein; Judith Lorenz; Nele Ruoff; Frank Hensel; Ines Beyer; Justus M Uller; Konrad Neukam; Bertram Illert; Matthias Eck; Hans Konrad M Uller-hermelink; H. Peter Vollmers

403

Characterisation of echidna IgM provides insights into the time of divergence of extant mammals  

Microsoft Academic Search

The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (C?) was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian C?s, and

Katherine Belov; Lars Hellman; Desmond W Cooper

2002-01-01

404

Fluorescent immunoassay visualization of sorbed pollutants  

SciTech Connect

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

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

1994-12-31

405

Gliadin Detection in Food by Immunoassay  

NASA Astrophysics Data System (ADS)

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

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

406

Aptamer-Au NPs conjugates-accumulated methylene blue for the sensitive electrochemical immunoassay of protein.  

PubMed

In this paper, we combine the advantages of aptamer, nanomaterial and antibody to design an electrochemical sandwich immunoassay for the ultrasensitive detection of human immunoglobulin E (IgE) by using methylene blue (MB) as electrochemical indicator. The sandwich structure is fabricated by using goat anti-human IgE as capturing probe. Aptamer-Au nanoparticles (NPs) conjugates are used both as a sandwich amplification element as well as an accumulation reagent of MB. Once the aptamer-Au NPs conjugates specifically bind to electrode surface, MB molecules are accumulated on its surface by the specific interaction of MB with G base of aptamer-Au NPs conjugates. Therefore, with the increase of human IgE concentration, more aptamer-Au-NPs conjugates are bound, and thus, more MB molecules are accumulated. A good linear relationship is obtained for the detection of human IgE over a range of 1-10,000 ng/ml with a lowest detection limit of 0.52 ng/ml. In addition, by using BSA, human IgA and human IgM as contrast, the excellent specificity of this sensing system for the detection of human IgE is also demonstrated. PMID:20188888

Wang, Jianlong; Munir, Ahsan; Li, Zhonghong; Zhou, H Susan

2010-04-15

407

B-1 cells in the bone marrow are a significant source of natural IgM.  

PubMed

Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies identified BM and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B-cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM-secreting cells in spleen and, for the first time, in the BM as IgM(+) IgD(lo/-) CD19(hi) CD43(+) CD5(+/-) B-1 cells. The newly identified population of BM B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and BM B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Overall, these data identify populations of non-terminally differentiated B-1 cells in spleen and BM as the most significant producers of natural IgM. PMID:22009734

Choi, Youn Soo; Dieter, Jacquelyn A; Rothaeusler, Kristina; Luo, Zheng; Baumgarth, Nicole

2012-01-01

408

The neonatally thymectomized rat: a model for compensatory IgM antibody formation in exocrine secretions.  

PubMed Central

The effect of T-cell deprivation on IgM antibodies in saliva was studied in rats. IgM was never detected in salivas obtained from normal or sham-thymectomized rats, but was detected in saliva samples from 8/15 (53.3%) rats that had undergone neonatal thymectomy. All (6/6) neonatally thymectomized rats exhibited an IgM antibody response to DNP in salivary secretions after local immunization with a T-dependent antigen (DNPBGG), while no IgA anti-DNP activity was detected in saliva from these antimals. IgM antibodies were detected in saliva from 5/7 thymectomized rats following local injection with a T-independent antigen (DNP-Lys-Gicoll). This was accompanied by detectable but substantially reduced levels of secretory IgA antibody in saliva from 7/7 rats. The results suggested that absent or decreased salivary IgA responses accompanying T-cell deprivation in the rats are compensated for by secretion of IgM antibodies into the saliva. The neonatally thymectomized rat may thus provide a model for the study of synthesis, secretion and protective potential of exocrine IgM antibodies. PMID:116959

Ebersole, J L; Taubman, M A; Smith, D J

1979-01-01

409

The role of IgM rheumatoid factor in experimental immune vasculitis.  

PubMed Central

The effect of IgM rhematoid factor (RF) on reversepassive cutaneous Arthus reaction in rats was studied. The RF was obtained from the serum cryoglobulin of a patient with symptoms of purpura, arthralgia and digital gangrene. The cryoglobulins was of IgG-IgM type and when given i.v it induced a prompt hypocomplementaemia in experimental animals. The purified RF also induced low serum complement levels when injected i.v. along with complexes of non-complement-fixing, aggregated IgG. A reverse passive Arthus reaction was induced by intradermal injection of IgG anti-bovine serum albumin (BSA), followed by an i.v. dose of antigen (Ag). The cutaneous inflammatory reaction was aggravated by simultaneous administration of IgM RF intradermally, but not by IgM without antibody (Ab) properties. Intradermal injection of low concentrations of non-complement-fixing IgG anti-BSA, along with normal human IgM, followed by i.v. injection of BSA, resulted in a complete lack of cutaneous inflammation. At higher Ab concentrations there was only a mild inflammation. However, when IgM RF was substituted for normal IgM and injected with non-complement-fixing anti-BSA, an effective reverse passive cutaneous Arthus reaction and vasculitis was induced. The inflammatory response was greatly suppressed by decomplementation of animals by cobra venom factor. This study provides evidence favouring an inflammatory, complement-dependent role for RF in vasculitis. PMID:157238

Floyd, M; Tesar, J T

1979-01-01

410

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

SciTech Connect

Immunoassay tests were used as in situ field screening tools for simultaneous assessment and remediation of soil contaminated with mercury and organics (polychlorinated biphenyls (PCB), polyaromatic hydrocarbons (PAH), and BTEX). Soil samples from approximately 200 sites including metering and compressor stations were investigated along gas pipelines. The suspected contamination originated from formerly used mercury manometers and pipeline liquids. An enzyme-linked immunosorbent assay (ELISA) was used for the detection of organic and mercury contaminants. ELISA combines selective antibodies with sensitive enzyme reactions to produce semiquantitative analytical systems capable of detecting very low levels of chemicals. Color sample tubes were compared to the color of standard tubes to semiquantitate the amount of mercury, PCB, PAH, and BBTEX present in the samples. Two standards were tested in order to eliminate the effects of false negatives (e.g., contaminant not detected when present) or false positives (e.g., contamination detected when not present). For verification purposes, selected samples that were determined to be below action level with the immunoassay tests were sent to the laboratory.

Hammes, U. [IT Corp., Houston, TX (United States)

1995-09-01

411

Application of Antibody and Fluorophore-Derivatized Liposomes to Heterogeneous Immunoassays for D-dimer  

E-print Network

Application of Antibody and Fluorophore-Derivatized Liposomes to Heterogeneous Immunoassays for D-type) immunoassay for the detection of thromboembolic disorders by assaying for d-dimer. D-dimer is the final. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate

Kilpatrick, Peter K.

412

Differentiation of classical swine fever virus infection from CP7_E2alf marker vaccination by a multiplex microsphere immunoassay.  

PubMed

Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E(rns), and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E(rns) antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E(rns) ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E(rns) antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals. PMID:25378351

Xia, Hongyan; Harimoorthy, Rajiv; Vijayaraghavan, Balaje; Blome, Sandra; Widén, Frederik; Beer, Martin; Belák, Sándor; Liu, Lihong

2015-01-01

413

Fluorimetric immunoassay for multianalysis of aflatoxins.  

PubMed

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

Kanungo, Lizy; Bhand, Sunil

2013-01-01

414

Differentiation of acute and chronic hepatitis B in IgM anti-HBc positive patients  

PubMed Central

AIM: To identify the factors that differentiate acute hepatitis B (AHB) from chronic hepatitis B with acute exacerbation (CHB-AE). METHODS: From 2004 to 2013, a total of 82 patients (male n = 52, 63.4%; female n = 30, 36.6%) with clinical features of acute hepatitis with immunoglobulin M antibodies to the hepatitis B core antigen (IgM anti-HBc) were retrospectively enrolled and divided into two groups; AHB (n = 53) and CHB-AE (n = 29). The AHB group was defined as patients without a history of hepatitis B virus (HBV) infection before the episode and with loss of hepatitis B surface antigen within 6 mo after onset of acute hepatitis. Biochemical and virological profiles and the sample/cutoff (S/CO) ratio of IgM anti-HBc were compared to determine the differential diagnostic factors. RESULTS: The multivariate analysis demonstrated that, the S/CO ratio of IgM anti-HBc and HBV DNA levels were meaningful factors. The S/CO ratio of IgM anti-HBc was significantly higher in the AHB group, while the HBV DNA level was significantly higher in the CHB-AE group. The optimal cutoff values of IgM anti-HBc and HBV DNA levels for differentiating the two conditions were 8 S/CO ratio and 5.5 log10 IU/mL, respectively. The sensitivity and specificity were 96.2% and 89.7% for the S/CO ratio of IgM anti-HBc and 81.1% and 72.4% for HBV DNA levels, respectively. The area under receiver operating characteristic curves of both the S/CO ratio of IgM anti-HBc and HBV DNA levels were not significantly different (0.933 vs 0.844, P = 0.105). When combining IgM anti-HBc and HBV DNA, the diagnostic power significantly improved compared to HBV DNA alone (P = 0.0056). The combination of these factors yielded a sensitivity and specificity of 98.1% and 86.2%, respectively. CONCLUSION: The combination of the S/CO ratio of IgM anti-HBc and HBV DNA levels was a useful tool for differentiating AHB from CHB-AE in patients with positive IgM anti-HBc.

Park, Ji Won; Kwak, Kyeong Min; Kim, Sung Eun; Jang, Myoung Kuk; Kim, Dong Joon; Lee, Myung Seok; Kim, Hyoung Su; Park, Choong Kee

2015-01-01

415

J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.  

PubMed

Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization. PMID:1736312

Randall, T D; Parkhouse, R M; Corley, R B

1992-02-01

416

Environmental monitoring by immunoassay: Current and future trends  

SciTech Connect

Immunoassays for low molecular weight molecules have become increasingly popular in part, this is a result of the simplicity and potentially low cost of immunoassays when compared with conventional analytical methods. Environmental application of immunoassays, however, poses new challenges. First, not only must the parent compound be detected but often one would also like to detect metabolites or environmentally weathered'' products. Monoclonal antibody technology offers the possibility of selecting antibodies that have specific binding characteristics, eg. those that bind to a family of similar compounds or to broad classes of chemicals. Next, a simple rapid sample preparation method must be available. Unlike clinical assays that run in serum or other biological fluids (ideal matrixes for antibodies), environmental sample consist of soils, sediments, chemical sludges, water and air samples. Thus, in all cases, with the possible exception of water samples, the analyte of interest must be extracted and presented to the antibody in an appropriate buffer system. Once these criteria are met, immunoassays present an attractive alternative for conventional analytical methods. The most direct application of immunoassays will undoubtedly as as screening assays, with positives being confirmed by gas liquid chromatography/mass spectrometry (GC/MS). However, coupling immunoassay with other analytical methods, such as with high-pressure liquid chromatography (HPLC) converts a single residue immunoassay into a multiresidue method. Finally, immunoaffinity chromatography procedures are useful as methods for sample clean-up, followed by immunochemical detection or more traditional detection methods. 15 refs., 6 figs., 2 tabs.

Stanker, L.H.; Watkins, B.E.; Vanderlaan, M.

1990-10-31

417

Enzyme Kinetics  

NSDL National Science Digital Library

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

Carl Stiefbold (University of Oregon; )

1998-01-01

418

Enzyme Kinetics.  

ERIC Educational Resources Information Center

Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

Moe, Owen; Cornelius, Richard

1988-01-01

419

Comparison of immunoassay screening tests and LC-MS-MS for urine detection of benzodiazepines and their metabolites: results of a national proficiency test.  

PubMed

For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity. PMID:23943436

Bertol, Elisabetta; Vaiano, Fabio; Borsotti, Maurizio; Quercioli, Massimo; Mari, Francesco

2013-01-01

420

Environmental Immunoassays: Alternative Techniques for Soil and Water Analysis  

USGS Publications Warehouse

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

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

1996-01-01

421

Immunoassay procedures for fiber optic sensors  

NASA Astrophysics Data System (ADS)

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

Ligler, Frances S.

1988-04-01

422

A new antibody in rheumatoid arthritis targeting glycated IgG: IgM anti- IgG-AGE  

Microsoft Academic Search

SUMMARY Hyperglycaemia and\\/or oxidative stress can cause IgG to be modified by advanced glycation end products (AGE). Three patients with aggressive rheumatoid arthritis (RA) and vasculitis are described who have high titres of IgM antibodies against AGE-modified IgG (IgM anti-IgG-AGE ). Diabetics and randomly selected patients with rheumatic diseases, including 50 additional RA patients, were tested for IgM and IgA

S. LIGIER; P. R. FORTIN; M. M. NEWKIRK

1998-01-01

423

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

Microsoft Academic Search

Summary 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

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

2002-01-01

424

Simple immunoassay for detection of PCBs in transformer oil.  

PubMed

A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed. PMID:16053103

Glass, Thomas R; Ohmura, Naoya; Taemi, Yukihiro; Joh, Takashi

2005-07-01

425

J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes.  

PubMed

Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM. Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies. PMID:2174785

Meng, Y G; Criss, A B; Georgiadis, K E

1990-11-01

426

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

Microsoft Academic Search

BACKGROUND: Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric\\/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that,

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

2004-01-01

427

Relationship Between Everolimus Blood Concentration Assessed Using the Innofluor Certican Fluorescence Polarization Immunoassay and the Architect i System Sirolimus Chemiluminescent Microparticle Immunoassay  

Microsoft Academic Search

Everolimus, an immunosuppressive macrolide derivative of sirolimus, has a narrow therapeutic index and variable bioavailability. Assessment of blood concentration of everolimus is necessary to improve immunosuppressive efficacy without increasing potential adverse effects. Recently, Seradyn, Inc (Indianapolis, Indiana) introduced a fluorescence polarization immunoassay (Innofluor Certican Fluorescent Polarization Immunoassay [FPIA]) for quantitation of everolimus blood concentration. This immunoassay has concentration-dependent cross-reactivity with

L. Coentrão; C. Carvalho; S. Sampaio; J. G. Oliveira; M. I. Pestana

2010-01-01

428

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

PubMed

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

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

2015-01-01

429

Enzyme Informatics  

PubMed Central

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

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