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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

A dot enzyme immunoassay for detection of IgM antibodies against phenolic glycolipid-I in sera from leprosy patients.  

PubMed

A visual dipstick dot enzyme immunoassay (EIA) for diagnosis of leprosy is described. The assay is based on detection of IgM antibodies against phenolic glycolipid (PGL-I) in sera from leprosy patients. The antigen (PGL-I or synthetic disaccharide of PGL-I) was dotted on a nitrocellulose pad stuck on a plastic strip (dipstick). Sera were used at a dilution of 1:200. Peroxidase coupled mouse anti-human IgM monoclonal antibodies were used as the conjugate. A positive test gave a blue dot against a white background. The test was highly specific for leprosy, and was quite sensitive for detection of bacilliferous (BL/LL) leprosy. The antigen dotted and preblocked dipsticks stored at room temperature upto 4 months of observation period, were unable in the assay. PMID:3543160

Kumar, S; Moudgil, K D; Band, A H; Naraynan, P R; Gupta, S K; Sharma, A K; Talwar, G P

1986-01-01

3

Enzyme Immunoassays for Measurement of Cytomegalovirus Immunoglobulin M Antibody  

PubMed Central

The diagnosis of congenital cytomegalovirus (CMV) infection is often accomplished by the detection of circulating antibody directed against CMV. We devised a method for measuring CMV-specific immunoglobulin M (IgM) based on the isolation of IgM antibody by reaction with a solid phase coated with antihuman IgM. The determination of IgM antibody specific for CMV was accomplished by the subsequent addition of CMV or control antigen and enzyme-labeled CMV antibody (solid phase-IgM method). We compared the sensitivity and specificity of this method with those of a conventional form of solid-phase enzyme immunoassay in which CMV antigen is bound to the solid phase (solid phase-antigen method). Both assay systems were capable of detecting CMV-specific IgM antibody in the sera of 10 babies with documented CMV infection and in those of the mothers of 4 of these babies. The solid phase-IgM method yielded negative results in all 66 sera available from babies who did not have congenital CMV infection. On the other hand, the solid phase-antigen system yielded false-positive results in 12 (18%) of these sera. In addition, the solid phase-antigen system yielded false-positive results in 8 of 12 sera obtained from patients with demonstrable rheumatoid factor. However, the solid phase-IgM system yielded negative results for the rheumatoid sera, provided that appropriate control reactions were performed. The solid phase-IgM system is thus a specific and sensitive method for the determination of CMV IgM antibody. PMID:6270191

Yolken, Robert H.; Leister, Flora J.

1981-01-01

4

Determination of anti-HBsAg IgM monoclonal antibodies in cell culture media by perfusion immunoassay.  

PubMed

A rapid, specific, perfusion immunoassay for active anti-HBsAg monoclonal IgM is described. The immunoassay requires less than 3.5 min per sample. The precision was found to be 3.6% at an IgM concentration of 17 microg/ml. A detection limit of 1 microg/ml IgM in culture media was determined. Assay results were found to correlate very well with standard size exclusion chromatography and radial immunodiffusion techniques. This perfusion immunoassay was demonstrated to be useful for determining anti-HBsAg IgM in complex matrices such as cell culture media. The utility of the immunoassay for monitoring production of anti-HBsAg IgM in a perfusion bioreactor is demonstrated. PMID:9005942

Brackett, J M; Cousineau, K L; Wang, H; Annapragada, A V; Cabal, O D; Gall, G S; Peterson, B; Robey, W G

1997-01-15

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.

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

1997-01-01

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. 19 figs.

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

1997-11-25

7

Fast dipstick dye immunoassay for detection of immunoglobulin G (IgG) and IgM antibodies of human toxoplasmosis.  

PubMed

A dipstick dye immunoassay (DDIA) was developed to detect immunoglobulin G (IgG) or IgM antibodies of toxoplasmosis infection in humans. The assays employ a blue colloidal dye particles (D-1) conjugated to sheep anti-human IgG and rabbit anti-human IgM as the visualizing agents and a soluble antigen of tachyzoites of Toxoplasma gondii strain RH (TSA) as the detective antigen. The mixture of dye-labeled anti-human antibody-special human antibody was captured by TSA onto a nitrocellulose membrane dipstick by means of immunochromatography. The assays are rapid (the whole test can be completed within 15 min), simple, and cheap, and they don't require any equipment. They are sensitive and specific for the detection of anti-Toxoplasma IgG or IgM antibodies and generally agree closely with the results from the enzyme-linked immunosorbent assay. The assays are especially suitable for field applications. PMID:15643007

Jin, Si; Chang, Zhu Yin; Ming, Xu; Min, Cao Li; Wei, He; Sheng, Liang You; Hong, Guan Xiao

2005-01-01

8

Advent of innovative chemiluminescent enzyme immunoassay.  

PubMed

Using 1,1'-oxalyldiimidazole (ODI) chemiluminescence detection, a new chemiluminescent enzyme immunoassay (CLEIA) was developed to quantify prostate specific antigen (PSA) in human serum. The results observed in ODI CLEIA were compared with those obtained in commercially available enzyme linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and luminol CLEIA. PSA complex-conjugated horseradish peroxidase (HRP) was formed from one-step sandwich immunoreaction of PSA, PSA primary antibody and PSA secondary antibody-conjugated HRP for 15 min in a strip-well at 36.5°C. CL substrate solution (Amplex Red and H2O2 in PBS buffer, pH 7.4) was added in the washed strip-well and incubated for 10 min at room temperature. When resorufin formed in this process was mixed with 1,1'-oxalyldi-4-methylimidazole and H2O2 in a testing tube, rapid and bright CL was observed. Detection limit (0.035 ng/ml) of PSA in ODI CLEIA was much lower than those (0.50 and 0.25 ng/ml) in commercially available ELISA and luminol CLEIA even though total incubation time of the former (25 min) was shorter than those of the latter (45 and 35 min). Also, the dynamic range (0-100 ng/ml, R2=0.9996) of ODI CLEIA was wider than those of other EIAs. In conclusion, the excellent correlation (r=0.9767) between ODI CLEIA and Advia Centaur XP Immunoassay System indicates that the accurate, precise, and rapid ODI CLEIA can be applied as a novel CLEIA capable of diagnosing and monitoring various diseases. PMID:20739174

Lee, Ji Hoon; Rho, Jee-Eun R; Rho, Tae-Ho D; Newby, John G

2010-10-15

9

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

PubMed Central

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

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

2013-01-01

10

Paramagnetic bead based enzyme electrochemiluminescence immunoassay for TNT  

Microsoft Academic Search

We have developed an electrochemiluminescence immunoassay system for TNT (2,4,6-trinitrotoluene) in which enzyme labelled antibodies bound to paramagnetic beads are concentrated on an electrode magnetically, and light emission is triggered electrochemically. Full details of the instrumentation used to carry out these immunoassays are described. The paramagnetic beads are coated with haptenylated dextrans prepared by substituting biotin and analogues of TNT

Robert Wilson; Charles Clavering; Alistair Hutchinson

2003-01-01

11

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...

12

Improvement of the lectin-antibody enzyme immunoassay of the alphafetoprotein carbohydrate chain for automation with the enzyme immunoassay robot.  

PubMed

The lectin-antibody enzyme immunoassay of the alphafetoprotein-L3 carbohydrate chain, a tumor marker of liver cancer, has not been automated. We improved the technique of the assay for automation. Consequently, alphafetoprotein-L3 and total alphafetoprotein were detected with two lectins using an automatic paramagnetic bead handling robot. This indicates that the improved method is potentially applicable to the automated enzyme immunoassay robot. PMID:16116296

Tamano, Koichi; Sugiura, Mika; Natsuki, Jun; Sawakami-Kobayashi, Kazumi; Tajima, Hideji; Machida, Masayuki

2005-08-01

13

Evaluation of microparticle enzyme immunoassays for immunoglobulins G and M to rubella virus and Toxoplasma gondii on the Abbott IMx automated analyzer.  

PubMed Central

The ability of the Abbott IMx automated analyzer to detect immunoglobulin G (IgG) and IgM antibodies to rubella virus and to Toxoplasma gondii was compared with the abilities of RUBAZYME, RUBAZYME-M, ABBOTT TOXO-G enzyme immunoassay, and ABBOTT TOXO-M enzyme immunoassay, respectively. Specimens that produced discordant results were evaluated by RUBACELL II, Behring Enzygnost-Rubella enzyme-linked immunosorbent assay, Behring Enzygnost Toxoplasmosis/IgG, and bioMerieux Toxo-ISAGA (immunosorbent agglutination assay), respectively. After resolution of discordant results, IMx Rubella IgG, IMx Rubella IgM, IMx Toxo IgG, and IMx Toxo IgM antibody assays had sensitivities of 99.9, 100, 98.0, and 100%; specificities of 98.9, 99.0, 97.5, and 98.7%; and accuracies of 99.8, 99.3, 97.8, and 98.8%, respectively. PMID:2681246

Schaefer, L E; Dyke, J W; Meglio, F D; Murray, P R; Crafts, W; Niles, A C

1989-01-01

14

Screening for treponemal infection by a new enzyme immunoassay.  

PubMed Central

A new enzyme immunoassay (EIA, Captia Syphilis-G) for detecting IgG antibodies against Treponema pallidum was evaluated as a screening test for syphilis. When serum samples were tested at a dilution of 1 in 20 (EIA20), the overall agreement between the IgG EIA and serological status based on the T pallidum haemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test was 99.2% (1310/1321). The sensitivity of the EIA20 was 98.4% (60/61) and the specificity 99.3% (1251/1260). Discrimination between patients with and without treponemal infection was good: the mean EIA20 absorbance ratios (patient/mean low titre positive control results) were 0.49 for antibody negative patients, 3.30 for patients with positive Venereal Diseases Research Laboratory (VDRL) test and TPHA results, and 1.77 for patients with negative VDRL but positive TPHA results. The cut off point for excluding treponemal infection was taken as 0.9. Specimens with ratios of more than 0.9 should be confirmed by the FTA-ABS test and evaluated for specific IgM antibodies to treponemes. When serum samples were tested at a 1 in 50 dilution (EIA50) the sensitivity was lower (80.3%) but the specificity was absolute. The reduction in sensitivity correlated with low absorbance ratios in the patients who were VDRL negative and TPHA positive. The screening performance of the IgG EIA20 is thus comparable with that provided by a combination of the VDRL test and TPHA. The potential for automation makes the EIA an attractive alternative, particularly in larger centres. Alternatively, the test can be performed at a 1 in 50 dilution (EIA50), at which level it is ideally suited for confirming the treponemal status of antibodies in serum samples preselected by positive cardiolipin antigen screening test results. PMID:2666302

Young, H; Moyes, A; McMillan, A; Robertson, D H

1989-01-01

15

Comparison of enzyme immunoassays for the diagnosis of bovine brucellosis  

Microsoft Academic Search

The indirect enzyme immunoassay for measurement of bovine antibody to Brucella abortus was tested on 15 716 Canadian sera to assess the specificity. These sera were also tested by the buffered plate antigen test. Two enzyme-linked immunosorbent assay (ELISA) formats were used for assessment of data: the targeting procedure using a positive control serum allowed to develop to an optical

K. H. Nielsen; L. Kelly; D. Gall; S. Balsevicius; J. Bosse; P. Nicoletti; W. Kelly

1996-01-01

16

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 ...

17

Evaluation of the Bartels Legionella Urinary Antigen enzyme immunoassay.  

PubMed

The Bartels Legionella Urinary Antigen enzyme immunoassay (Intracel, USA) is intended for the presumptive diagnosis of past or current Legionnaires' disease by qualitative detection of Legionella pneumophila serogroup 1 antigen in human urine. This test was evaluated using single urine samples collected from 349 patients with lower respiratory tract infection of known aetiology. Specificity was estimated as 100% (181 samples, 95% CI: 98%-100%); sensitivity for Legionella pneumophila serogroup 1 was 98.8% (167 samples, 95% CI: 95.7%-99.9%). Assessing assay results using a Visual Interpretation Card provided by the manufacturer in place of a photometer gave rise to one false-positive result among the 78 control samples examined. Providing the endpoint of this assay is determined photometrically, the Bartels Legionella Urinary Antigen enzyme immunoassay appears to be a highly specific and sensitive kit for the diagnosis of infection caused by Legionella pneumophila serogroup 1. PMID:11757977

Harrison, T G; Doshi, N

2001-10-01

18

An indirect enzyme immunoassay for the mycotoxin citrinin.  

PubMed Central

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%. PMID:7646038

Abramson, D; Usleber, E; Märtlbauer, E

1995-01-01

19

Inter?Test Comparison Between Filter Paper Absorbed Blood Eluate and Serum for Malaria Serology by Enzyme Immunoassay: An Operational Feasibility  

Microsoft Academic Search

Antimalarial IgG and IgM were detected by enzyme immunoassay in finger?stick blood samples collected in capillary tubes and also spotted onto Whatman filter paper. Assay was performed in 92 blood samples obtained from 53 falciparum malaria patients, representing 23 fever cases (malaria negative) and 16 healthy individuals. A simple indirect ELISA was done using Plasmodium falciparum lysate and MSP119 peptide

Sukla Biswas

2005-01-01

20

Enzyme immunoassay for screening of sulfathiazole in honey.  

PubMed

A simple enzyme immunoassay (EIA) was developed to screen honey samples for sulfathiazole (ST) adulteration. Honey samples required only a 30-fold dilution before use in the procedure. Because 96 well microtiter plates were used and only 100 microL of diluted honey sample was required per well, numerous replicates or samples could be tested simultaneously. The EIA was able to detect at least 0.3 ppm levels of ST in honey and also provide a rough quantitation of ST amounts. PMID:2289917

Sheth, H B; Sporns, P

1990-01-01

21

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.  

PubMed

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection. PMID:1354900

Bignami, G S; Raybould, T J; Sachinvala, N D; Grothaus, P G; Simpson, S B; Lazo, C B; Byrnes, J B; Moore, R E; Vann, D C

1992-07-01

22

Performance of a Cytomegalovirus IgG Enzyme Immunoassay Kit Modified To Measure Avidity  

PubMed Central

The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity. PMID:24671557

Lapé-Nixon, Mary; Novak-Weekley, Susan M.

2014-01-01

23

A plasmonic nanosensor for immunoassay via enzyme-triggered click chemistry.  

PubMed

Current techniques for plasmonic immunoassay often require the introduction and additional conjugation of enzyme, and thus cannot accommodate conventional immunoassay platforms. Herein, we develop a plasmonic nanosensor that well accommodates conventional immunoassays and dramatically improves their sensitivity and stability. This plasmonic nanosensor directly employs alkaline phosphatase-triggered click chemistry between azide/alkyne functionalized gold nanoparticles as the readout. This straightforward approach broadens the applicability of nanoparticle-based immunoassays and has great potential for applications in resource-constrained settings. PMID:25423357

Xianyu, Yunlei; Wang, Zhuo; Jiang, Xingyu

2014-12-23

24

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

25

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

26

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

PubMed

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

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

2014-04-01

27

Detection of rat, porcine, and bovine group B rotavirus in fecal specimens by solid-phase enzyme immunoassay.  

PubMed Central

An enzyme immunoassay that uses easily regenerated reagents was developed and evaluated for the ability to detect group B rotaviruses (GBR) in fecal specimens. Homologous rat GBR and heterologous porcine and bovine GBR were detected by this immunoassay, although a human GBR isolate was not. This immunoassay should prove useful in studies of GBR infections of animals. PMID:8027324

Vonderfecht, S L; Lindsay, D A; Eiden, J J

1994-01-01

28

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

29

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

PubMed Central

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

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

2014-01-01

30

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

PubMed Central

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

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

1991-01-01

31

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

32

Comparison of five enzyme immunoassays, electron microscopy, and latex agglutination for detection of rotavirus in fecal specimens.  

PubMed Central

Five different enzyme immunoassays, electron microscopy, and latex agglutination (Slidex; bioMerieux) were compared for the rapid detection of human rotavirus in fecal specimens. The enzyme immunoassay using rotavirus polyclonal antiserum (Dakopatts) with simple in-house modifications was shown by the use of confirmatory tests to be the most sensitive and specific procedure. PMID:2536758

Kok, T W; Burrell, C J

1989-01-01

33

Evaluation of the Roche Elecsys Toxo IgG and IgM electrochemiluminescence immunoassay for the detection of gestational Toxoplasma infection.  

PubMed

Unidentified gestational infection with Toxoplasma gondii may lead to fetal infection with severe complications later in childhood. Because diagnosis of maternal infection solely depends on serology, routine tests with high sensitivity and specificity are required. In this study, the new Roche Elecsys Toxo IgG and IgM immunoassay was compared with Sabin-Feldman dye test and immunosorbent agglutination assay-IgM as reference test. Serum samples were analyzed from 927 pregnant women, including 100 negative, 706 chronic, and 121 acute infections. The combination of both Elecsys IgG and IgM assays demonstrated high sensitivity and specificity of 97.1% and 100.0%, respectively, and a positive and negative predictive value of 100.0% and 81.3%, respectively. The Elecsys assay is a useful tool as a first-line screening method to detect gestational infections. However, if gestational infection is assumed, confirmatory testing by a reference laboratory might be necessary to discriminate between pre- and postconceptional infection to start antiparasitic treatment to avoid mother-to-fetus transmission and severe sequelae. PMID:20884150

Prusa, Andrea-Romana; Hayde, Michael; Unterasinger, Lukas; Pollak, Arnold; Herkner, Kurt R; Kasper, David C

2010-12-01

34

Analytica Chimica Acta 466 (2002) 247256 Development of an enzyme immunoassay for  

E-print Network

Analytica Chimica Acta 466 (2002) 247­256 Development of an enzyme immunoassay for linoleic acid-linked immunosorbent assay for the diol derivatives of linoleic acid, cis-9,10-dihydroxyoctadec-12(Z)-enoic acid synthesized by conjugation of LTXD/iso-LTXD, dihydroxystearic acid, ricinoleic acid (OLE), ricelaidic acid

Hammock, Bruce D.

35

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...

36

Design of liquid level measurement for sampling module of full-automatic enzyme immunoassay instrument  

Microsoft Academic Search

To support the need for the design of sampling module in the full-automatic enzyme immunoassay instrument, this paper completed the design and verification of liquid level detector based on capacitive sensor. Using the capacitance effect existing between the sample needle and the ground (the machine frame), we take the sample needle and the ground as the capacitor poles. By means

Hong Li; Lianqing Zhu; Haitao Chang; Yan Bao

2010-01-01

37

Enzyme Immunoassay for the Quantification of Mitomycin C Using ß-Galactosidaseas a Label1  

Microsoft Academic Search

A mitomycin C (MMC) antibody was produced following immunization of rabbits with a MMC-bovine serum albumin conjugate, which was newly synthesized by coupling MMC to mercaptosuccinylated bovine serum albumin via a cross-linker, W-maleoyl aminobutyric acid. Enzyme labeling of MMC was performed using \\/S-o-galactosidase (EC 3.2.1.23) via m-ma- leoyl benzoic acid. An enzyme immunoassay for MMC was developed utilizing these reagents

Kunio Fujiwara; Hitoshi Saikusa; Motomi Yasuno; Tsunehiro Kitagawa

38

Lectin-enzyme immunoassay of transferrin sialovariants using immobilized antitransferrin and enzyme-labeled galactose-binding lectin from Ricinus communis.  

PubMed

A heterologous lectin-enzyme immunoassay is described. Microtiter plate wells were coated with affinity-purified antibodies to human transferrin. After incubation with transferrin sialovariants, prepared by limited neuraminidase treatment and separated with chromatofocusing, a lectin-enzyme-streptavidin complex was added. A good correlation was obtained between the number of terminal galactose groups on transferrin and the response in the lectin-enzyme immunoassay using Ricinus communis agglutinin as the galactose-binding lectin. The results indicate that characterization of glycosylation is possible with less than a microgram of the glycoprotein available, using lectin-enzyme immunoassays. PMID:3322101

Pekelharing, J M; Vissers, P; Peters, H A; Leijnse, B

1987-09-01

39

Inter-test comparison between filter paper absorbed blood eluate and serum for malaria serology by enzyme immunoassay: an operational feasibility.  

PubMed

Antimalarial IgG and IgM were detected by enzyme immunoassay in finger-stick blood samples collected in capillary tubes and also spotted onto Whatman filter paper. Assay was performed in 92 blood samples obtained from 53 falciparum malaria patients, representing 23 fever cases (malaria negative) and 16 healthy individuals. A simple indirect ELISA was done using Plasmodium falciparum lysate and MSP1(19) peptide as antigens. Total IgG and IgM contents were also estimated in individual sera and filter paper eluate by single radial immunodiffusion (SRID). Assay results of both serum and filter paper eluates were compared. The sensitivity and specificity of the assays for IgG measurement were comparable between serum and filter paper eluates (P < 0.001), whereas, in case of IgM, detection level was poor in filter paper eluates as observed by ELISA and SRID. The filter paper eluates may serve the purpose of antigen-specific IgG detection in seroepidemiological surveys. PMID:15552593

Biswas, Sukla

2004-01-01

40

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

PubMed

A sensitive and reproducible high-performance liquid chromatographic (HPLC) method is described for assay of gentamicin in serum using ultrafiltration of serum samples and pre-column derivatization with o-phthalaldehyde. The procedure was compared with a bioassay and an enzyme immunoassay. Coefficients of correlation were 0.98 for HPLC vs. bioassay and 0.97 for HPLC vs. immunoassay. The between-day imprecision (n = 5) was estimated from the coefficients of variation at a concentration of 6 mg/l. Values were 9.4% for the bioassay, 6.5% for the immunoassay and 2.4% for the chromatographic method. The major advantage of the method described is simplified sample preparation and optimal serum extraction. The latter is important in routine use because it prolongs column life and thus reduces costs. PMID:6761112

Essers, L

1982-12-01

41

Comparative Analysis of Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay Using Virus-Like Particles or Virus-Infected Mouse Brain Antigens To Detect IgM Antibody in Sera from Patients with Evident Flaviviral Infections  

Microsoft Academic Search

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source

Derek A. Holmes; David E. Purdy; Day-Yu Chao; Amanda J. Noga; Gwong-Jen J. Chang

42

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

43

Design of liquid level measurement for sampling module of full-automatic enzyme immunoassay instrument  

NASA Astrophysics Data System (ADS)

To support the need for the design of sampling module in the full-automatic enzyme immunoassay instrument, this paper completed the design and verification of liquid level detector based on capacitive sensor. Using the capacitance effect existing between the sample needle and the ground (the machine frame), we take the sample needle and the ground as the capacitor poles. By means of the RC oscillation circuit, we convert the change of capacitance to frequency for a measure, in order to achieve the level detection. The stepping motor controls the sample arm with the modular design, and C8051F330 is chosen as main control chip in this system. Through the experimental analysis, we proved the reliability of the detection of the sample level based on the capacitance liquid level detection principle. This study provided a theoretical basis to the level parameters of the full-automatic enzyme immunoassay instrument.

Li, Hong; Zhu, Lianqing; Chang, Haitao; Bao, Yan

2011-05-01

44

Design of liquid level measurement for sampling module of full-automatic enzyme immunoassay instrument  

NASA Astrophysics Data System (ADS)

To support the need for the design of sampling module in the full-automatic enzyme immunoassay instrument, this paper completed the design and verification of liquid level detector based on capacitive sensor. Using the capacitance effect existing between the sample needle and the ground (the machine frame), we take the sample needle and the ground as the capacitor poles. By means of the RC oscillation circuit, we convert the change of capacitance to frequency for a measure, in order to achieve the level detection. The stepping motor controls the sample arm with the modular design, and C8051F330 is chosen as main control chip in this system. Through the experimental analysis, we proved the reliability of the detection of the sample level based on the capacitance liquid level detection principle. This study provided a theoretical basis to the level parameters of the full-automatic enzyme immunoassay instrument.

Li, Hong; Zhu, Lianqing; Chang, Haitao; Bao, Yan

2010-12-01

45

Comparison of glucan detection and galactomannan enzyme immunoassay in gastrointestinal and systemic murine candidiasis  

Microsoft Academic Search

Mouse models of systemic and gastrointestinal infection with the yeast Candida albicans were used to investigate the ability of a commercial mannan antigen enzyme immunoassay and a commercial (1?3) ?-D-Glucan limulus assay to detect systemic infection and to differentiate between colonization and infection. Both assays were positive in all i.v. infected mice and negative in all uninfected control mice. In

Thomas Nichterlein; Dieter Buchheidt; Andreas Hein; Klaus-Peter Becker; Korinna Mosbach; Marianne Kretschmar

2003-01-01

46

Novel Recombinant-Antigen Enzyme Immunoassay for Serological Diagnosis of Syphilis  

Microsoft Academic Search

Enzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinant Treponema pallidum antigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the native T. pallidum antigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis

H. YOUNG; A. MOYES; L. SEAGAR; A. MCMILLAN

1998-01-01

47

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

48

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

49

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

PubMed Central

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

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

2013-01-01

50

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

51

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

PubMed

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

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

2011-01-01

52

Application of enzyme immunoassays for the confirmation of clinically suspect plague in Namibia, 1982.  

PubMed

An outbreak of plague occurred in Ovamboland, northern Namibia, late in 1982. Blood cultures, sera and blood clots were tested to obtain laboratory confirmations for clinically suspect cases of the disease. Isolation of the bacillus (Yersinia pestis) was attempted from blood cultures; sera were tested for antibody by passive haemagglutination (PHA) and enzyme-linked immunosorbent assay (ELISA). Sera and clots also were tested by ELISA for the specific F1 plague antigen. All the ELISA procedures were based on a monoclonal antibody to F1 antigen to ensure specificity.Thirty-eight cases were confirmed as plague: 50% by isolation, 34% by antibody responses, and 16% by the detection of antigenaemia. All isolates of Y. pestis were capable of producing F1 antigen, and significant antibody responses were observed in bacteriologically confirmed cases with paired sera. Patients who experienced sero-conversion had a higher IgM titre than IgG titre during the first nine days of hospitalization, while patients hospitalized for 17 or more days had IgG titres that were higher than the IgM titres. The relationship between IgM and IgG antibody titres is discussed with reference to identifying very recent infections. PHA titres increased and declined with IgM titres but were lower and more transient.ELISA procedures increased laboratory confirmations of plague by 23% above the numbers achieved using blood cultures and PHA tests alone. The ELISA to detect F1 antigen accounted for 86% of this increase by confirming cases where bacteriological isolation was not done. This ELISA did not replace the requirement for bacteriological isolation, since seven bacteraemic patients did not demonstrate antigenaemia. PMID:3492308

Williams, J E; Arntzen, L; Tyndal, G L; Isaäcson, M

1986-01-01

53

Development of a sensitive enzyme immunoassay for the determination of vinpocetine in human plasma.  

PubMed

An Enzyme immunoassay for the quantitative determination of vinpocetine (CAS-42971-09-5) in human plasma has been developed. The lower limit of quantification is 0.1 ng/ml plasma. The assay shows no cross reactivity with the major metabolite apovincaminic acid. Because of a strong unspecific binding of vinpocetine to plasma proteins an extraction step was necessary. The inter- and intra-assay reproducibility of the test (coefficient of variation) is in a range of 1.1 and 18.3%. PMID:1472136

Reck, B; Dingler, E; Lohmann, A

1992-10-01

54

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

55

Development of a simple enzyme immunoassay for the determination of ovine luteinizing hormone.  

PubMed

The present study describes the development and validation of a simple sensitive and specific sandwich enzyme immunoassay (EIA) for the quantification of ovine luteinizing hormone (LH) in plasma. Microtitre plates were coated with the capture antibody 518b7 anti-bovine LH. A second peroxidase-labelled anti-ovine LH antibody was used as tracer. A simple 3-step procedure was used for the sample analysis; (1) incubation of standards and samples with the pre-coated antibody plates for 2 h at 37 degrees C; (2) incubation with the peroxidase-labelled antibody for 1 h at room temperature; and (3) colour development with TMB substrate. A linear dose-response curve was obtained in the range 0-10 ng/ml (r2 > 0.99). The detection limit was 0.05 ng/ml, and the intra-assay and inter-assay coefficients of variation were 7% and 11.7%, respectively. The theoretical stability of microplates and reagents was calculated, this being greater than one year. Low or undetectable cross-reactivities were recorded for follicle-stimulating hormone, bovine thyroid-stimulating hormone, equine chorionic gonadotrophin and a gonadotrophin-releasing hormone (GnRH) analogue. The EIA was biologically validated by the determination of plasma LH concentrations of nine Rasa Aragonesa ovariectomized and estradiol-implanted ewes after a double GnRH challenge. In conclusion, this enzyme immunoassay provides an efficient, simple and sensitive method for the routine analysis of ovine LH. PMID:17225084

Valares, J A; Abecia, J A; Forcada, F; Palacín, I; Mata, L; Razquin, P

2007-05-01

56

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

57

Cross-Reactivity of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Cryptococcus Species in the Commercial Platelia Aspergillus Enzyme Immunoassay?  

PubMed Central

Cross-reactivity in the Platelia Aspergillus enzyme immunoassay was evaluated using 120 sera from patients with paracoccidioidomycosis, histoplasmosis, and cryptococcosis. At a cutoff value of 0.5, positivity rates were 50%, 67%, and 50%, respectively. The implications for these findings are discussed. PMID:19020109

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

2009-01-01

58

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

E-print Network

for the Analysis of Atrazine in Water Gerry J. Reimer,,§ Shirley J. Gee, and Bruce D. Hammock*, CanTest, Ltd of California, Davis, California 95616 Immunoassays for atrazine based on a time-resolved fluorescent label and an enzyme label were optimized and utilized to measure atrazine in water. The time-resolved fluorescent

Hammock, Bruce D.

59

Clinical immunoassay instrument markets  

SciTech Connect

The present status and future prospects of the market for clinical immunoassay instruments is discussed. The market shares for the five basic instrument types - nephelometric immunoassay, fluorescence immmunoassay, enzyme immunoassay, luminescence immunoassay, and radioimmunoassay are presented. It is noted that radioimmunoassay hold a major, but decreasing, share of the market.

Not Available

1984-11-01

60

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

61

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

62

Detection of immunoglobulin G antibodies to Helicobacter pylori in urine by an enzyme immunoassay method.  

PubMed Central

Urine and serum samples from 306 patients undergoing upper endoscopy were evaluated prospectively to determine the presence of immunoglobulin G (IgG) antibodies to Helicobacter pylori by an enzyme immunoassay method. Forty-nine selected urine specimens were also tested by Western blotting (immunoblotting). When compared with bioptic methods (culture, stain, urease testing), the sensitivity and specificity of the assay for urine IgG to H. pylori were 95.9 and 90%, respectively. Results of testing of serum and urine for IgG to H. pylori were concordant for 95% of samples. Western blot analysis revealed a highly variable antibody response to H. pylori antigens among patients. Detection of IgG antibody to H. pylori in urine is simple and reflects the presence or absence of H. pylori infection. PMID:8370747

Alemohammad, M M; Foley, T J; Cohen, H

1993-01-01

63

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

PubMed

New rapid method of lateral flow enzyme immunoassay (LFEIA) for progesterone detection in whole cows' milk was developed. The test system utilized horseradish peroxidase as a label along with the substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain an insoluble blue colored product of the enzyme reaction on a surface of analytical membrane (test and control lines). Several aspects of LFEIA were optimized: time of the signal detection, membrane materials and assay conditions. Resulting competitive LFEIA can be performed within 15minutes with the limit of progesterone detection of 0.8ng/ml. Progesterone concentration in whole milk samples was determined by LFEIA and enzyme-linked immunosorbent assay (ELISA). The results obtained were in good correlation (R=0.97, n=46). Thus new sensitive LFEIA can be successfully used for on-site monitoring of oestrus status of cows' reproductive system and for early none-pregnancy detection. The method is fast, easy to perform and needs no preliminary sample preparation. PMID:25476365

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

2015-01-15

64

Rapid chemiluminescent sandwich enzyme immunoassay capable of consecutively quantifying multiple tumor markers in a sample.  

PubMed

Using the role of p-iodophenol in enzyme assay, enhanced 1,1'-oxalyldiimidazole chemiluminescent enzyme immunoassays (ODI-CLEIAs) were developed to consecutively quantify trace levels of triple tumor markers, such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) in a sample. Due to the high sensitivity of enhanced ODI-CLEIAs, it was possible to fix the incubation times (1) to capture a tumor marker with two antibodies, which are primary antibody immobilized on the surface of polystyrene strip-well and detection antibody-conjugated horseradish peroxidase (HRP), and (2) to form resorufin with the addition of substrates (e.g., Amplex Red, H2O2) in order to quantify triple markers in human serum. Enhanced ODI-CLEIAs capable of consecutively and rapidly quantifying triple markers with the same incubation time were more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) capable of separately and slowly quantifying them with different incubation times. In addition, accuracy, precision, and recovery of enhanced ODI CLEIAs in the presence of p-iodophenol were acceptable within statistical error range. PMID:25127571

Kim, Julie; Kim, Jennie; Rho, Tae Ho D; Lee, Ji Hoon

2014-11-01

65

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

PubMed

A novel and ultrasensitive chemiluminescence immunoassay (CLIA) method based on multiple enzyme layers assembled multiwall carbon nanotubes (MWCNTs) as signal amplification labels was developed by employing luminol-H(2)O(2)-HRP-bromophenol blue (BPB) enhanced chemiluminescence (CL) system for the detection of a cancer biomarker in human serum samples, as exemplified by the measurement of alpha-fetoprotein (AFP) as a model protein. In this study, horseradish peroxidase (HRP) was assembled onto MWCNTs templates layer-by-layer (LBL) through electrostatic interactions with polyion PDDA, and further conjugated with AFP secondary antibodies (Ab(2)) as the enzyme label. The resulting LBL assembly could maximize the ratio of HRP/Ab(2) which could amplify the sensitivity greatly. To the best of our knowledge, it was the first time for this strategy applied in CLIA to date. Under the optimum conditions of luminol-H(2)O(2)-HRP-BPB CL system and the sandwich immunoreactions, a linear range from 0.02 to 2.0 ng/mL (R=0.9980) was obtained with the detection limit of 8.0 pg/mL (3sigma) which was two orders of magnitude lower than standard ELISA method. Furthermore, accurate detection of AFP in human serum samples was also demonstrated by comparison to ELISA assays. From the above results, such signal amplification strategy proposed by the novel CNT-LBL enzyme label showed an excellent promise for ultrasensitive detection of cancer biomarkers in clinical laboratory. PMID:19345084

Bi, Sai; Zhou, Hong; Zhang, Shusheng

2009-06-15

66

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

PubMed

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

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

1995-07-01

67

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

PubMed

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

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

2012-10-01

68

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

NASA Astrophysics Data System (ADS)

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

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

1993-03-01

69

Comparison of enzyme immunoassay–based assays for environmental Alternaria alternata  

PubMed Central

Background Alternaria alternata–derived allergenic materials are causes of human disease. Several immunoassays exist to quantify these materials. Objective To compare methods for evaluating Alternaria content. Methods Four methods, including 1 monoclonal antibody (MAb)–based assay specific for recombinant Alt a 1, 1 MAb-based assay for chromatographically purified Alt a 1, 1 polyclonal antibody (PAb)–based assay for chromatographically purified Alt a 1, and 1 PAb-based assay for whole Alternaria extract, were evaluated. Environmental samples collected as part of the National Survey of Lead and Allergens in Housing were examined. Alternaria spore counts were determined in dust by observation. Results The MAb-based assay for recombinant Alt a 1 detected Alternaria in few samples (25%); the PAb-based assay for whole Alternaria proteins detected antigen in 97% of the samples. The PAb- and MAb-based assays for purified Alt a 1 detected antigen in 100% of the samples. There was a significant positive correlation between the 2 assays directed against purified Alt a 1. There was a positive correlation between the PAb-based assay for whole Alternaria and the PAb-based assay for Alt a 1. Nearly all the dust samples contained Alternaria spores, and there was a strong positive correlation between counts and all assays. Conclusion Because of the multifaceted nature of Alternaria, the disparities between methods for quantifying Alternaria, the cross-reactivity between fungal allergens, and the documented genetic promiscuity of this fungus, enzyme immunoassays using PAbs against a range of Alternaria proteins will probably produce the most reliable estimation of overall Alternaria exposure in house dust. PMID:17042141

Barnes, Charles; Portnoy, Jay; Sever, Michelle; Arbes, Samuel; Vaughn, Ben; Zeldin, Darryl C.

2007-01-01

70

[Diagnostic efficiency of excretory-secretory Trichinella antigens in enzyme immunoassay].  

PubMed

The investigation deals with the enzyme immunodiagnosis of trichinosis in some mammalian species involved in the circulation of this widespread menacing anthropozoonosis in the natural synanthropic foci in the Russian Federation. The use of T. spiralis immunodominant antigens of a molecular mass of 63-29 kDa as enzyme immunoassay (EIA) diagnostic kits for the lifetime diagnosis may ensure a rather effective lifetime detected of the Trichinella-infested omnivores and carnivores (pigs and cats) participating in the circulation of the causative agent in the synanthropic foci. Also, these antigens are suitable for the study of the immunological aspects of tricinosis when T. spiralis invasion is simulated on laboratory animals (rodents). False-negative EIAs are associated with the specific features of immunological responsiveness of each organism, the degree of infectivity (immunological tolerance in intensive inoculation), the stage of invasion, the biological characteristics of a helminth (such as the immunosuppressive effect of the parasite on the host during larval migration), rather than with the quality of the used components of a response. PMID:18274147

Odoevskaia, I M

2007-01-01

71

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

PubMed Central

Soluble exoantigens in the supernatants of Babesia bovis cultures have been shown to be efficient immunogens against bovine babesiosis. We used a two-site enzyme immunoassay to monitor the release of these antigens during in vitro cultivation. Bovine immunoglobulin G was isolated from serum of an adult cow previously immunized with culture-derived B. bovis exoantigens and challenged via needle with virulent parasites. The specific immunoglobulin G was used as a capture antibody and as an enzyme-conjugated recognizing antibody. The optimal protein concentration of capture antibody was 10 micrograms/ml. The 24-h cultures showed the greatest antigen concentration. The test was sensitive for detection of differences in species-specific antigenic activity among B. bovis isolates, for determining loss of antigenicity during storage and formalinization, and for monitoring the kinetics of exoantigen release during in vitro cultivation. Antigens cross-reactive with the other major Babesia species of cattle, Babesia bigemina, were also detected with this assay. The high specificity, sensitivity, and reproducibility of this technique should facilitate detection and quantitation of Babesia antigens during purification and in standardization of candidate immunogens. PMID:3308949

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

1987-01-01

72

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

PubMed Central

Campylobacter spp. are fastidious microorganisms, and their detection by culture depends on the freshness of the stool sample and the skills of the laboratory staff. To improve laboratory diagnosis, assays for the detection of specific antigens have been developed. Here, we evaluated two assays for the detection of Campylobacter spp.-specific antigens, i.e., one immunochromatographic test and one enzyme-linked immunosorbent assay (EIA), in 38 frozen Campylobacter spp.-positive specimens and prospectively in 533 fresh stool samples with a conventional enzyme immunoassay (EIA) and culture. Both assays were positive for 36 samples with Campylobacter jejuni and one with Campylobacter coli among 38 Campylobacter spp.-positive frozen samples. One Campylobacter lari-positive sample was identified by the immunochromatographic assay (ICA) only. In a prospective study performed within the course of routine microbiology, both assays were positive for 24/25 C. jejuni culture-positive samples (positive percent agreement, 96.0% [95% CI: 78.9–100%]). ICA and EIA also were positive for 14 and 10 culture-negative samples, respectively (negative percent agreement: ICA, 97.2% [95% CI: 95.4–98.4%]; EIA, 98.0% [95% CI: 96.4–99.0%]). In conclusion, the high agreement between both antigen-detection assays and culture indicates that both assays may be initially performed followed by culture only upon a positive test result. PMID:25215191

Ignatius, Ralf

2014-01-01

73

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

PubMed

Campylobacter spp. are fastidious microorganisms, and their detection by culture depends on the freshness of the stool sample and the skills of the laboratory staff. To improve laboratory diagnosis, assays for the detection of specific antigens have been developed. Here, we evaluated two assays for the detection of Campylobacter spp.-specific antigens, i.e., one immunochromatographic test and one enzyme-linked immunosorbent assay (EIA), in 38 frozen Campylobacter spp.-positive specimens and prospectively in 533 fresh stool samples with a conventional enzyme immunoassay (EIA) and culture. Both assays were positive for 36 samples with Campylobacter jejuni and one with Campylobacter coli among 38 Campylobacter spp.-positive frozen samples. One Campylobacter lari-positive sample was identified by the immunochromatographic assay (ICA) only. In a prospective study performed within the course of routine microbiology, both assays were positive for 24/25 C. jejuni culture-positive samples (positive percent agreement, 96.0% [95% CI: 78.9-100%]). ICA and EIA also were positive for 14 and 10 culture-negative samples, respectively (negative percent agreement: ICA, 97.2% [95% CI: 95.4-98.4%]; EIA, 98.0% [95% CI: 96.4-99.0%]). In conclusion, the high agreement between both antigen-detection assays and culture indicates that both assays may be initially performed followed by culture only upon a positive test result. PMID:25215191

Regnath, Thomas; Ignatius, Ralf

2014-09-01

74

Enzyme immunoassay for phycocyanin as the main component of spirulina color in foods.  

PubMed

An enzyme immunoassay (EIA) for phycocyanin in foods was developed. Anti-phycocyanin monoclonal antibodies were obtained from A/J mice immunized with phycocyanin. The phycocyanin in a food was extracted by dissolving the sample in a borate buffer solution, pH 8.0 (BBS) and adjusting the pH value of this solution to 8.0 with NaOH. The extract was then diluted more than 10 fold with 1% gelatin in BBS. Phycocyanin was determined by avidin-biotin sandwich EIA, using the P26-8 monoclonal antibody as the solid-phase antibody and the P277-4 monoclonal antibody as the enzyme-labeled antibody. The working range for a quantitative analysis was 100-1000 ng/ml, and the detection limit was 10 micrograms/g of the original sample. Recoveries of phycocyanin from foods by this assay were > 71% for candy, and > 66% for ice cream and sherbet. Phycocyanin was assayed in 22 blue-, green-, purple-, and brown-colored commercial foods, and detected in one green colored-jelly at 49 micrograms/g. PMID:8824825

Yoshida, A; Takagaki, Y; Nishimune, T

1996-01-01

75

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

76

On-Chip Enzyme Quantification of Single Escherichia coli Bacteria by Immunoassay-based Analysis.  

PubMed

Individual bacteria of an isogenic population can differ significantly in their phenotypic characteristics. This cellular heterogeneity is thought to increase the adaptivity to environmental changes on a population level. Analytical methods for single-bacteria analyses are essential to reveal the different factors that may contribute to this cellular heterogeneity, among them the stochastic gene expression, cell cycle stages and cell aging. Although promising concepts for the analysis of single mammalian cells based on microsystems technology were recently developed, platforms suitable for proteomic analyses of microbial cells are by far more challenging. Here, we present a microfluidic device optimized for the analysis of single Escherichia coli bacteria. Individual bacteria are captured in a trap and isolated in a volume of only 155 pL. In combination with an immunoassay-based analysis of the cell lysate, the platform allowed the selective and sensitive analysis of intracellular enzymes. The limit of detection of the developed protocol was found to be 200 enzymes. Using this platform, we could investigate the levels of ?-galactosidase in cells grown under different nutrient conditions. We successfully determined the enzyme copy numbers in cells cultured in defined medium (3517 ± 1578) and in complex medium (4710 ± 2643), and verified the down-regulation of expression in medium that contained only glucose as carbon source. The strong variations we found for individual bacteria confirm the phenotype heterogeneity. The capability to quantify proteins and other molecules in single bacterial lysates is encouraging to use the new analysis platform in future proteomics studies of isogenic bacteria populations. PMID:25409480

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

2014-12-16

77

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

78

A Competitive Enzyme-Linked Immunoassay for the Quantitation of Soluble Human Immunoglobulin G and Albumin in Nanogram Amounts  

Microsoft Academic Search

SummaryBackground: Some clinical and experimental questions require quantitation of soluble serum proteins in amounts lower than those which are detectable by radial immunodiffusion (RID) or nephelometry. Materials and Methods: We describe a competitive enzyme-linked immunoassay (CELIA) for the quantitation of minor amounts of soluble human IgG and albumin. Results: The assay enables the detection of IgG and albumin in the

B. K. Flesch; K. Landmann; I. Oschlies; M. Wunderlich; J. Neppert

1997-01-01

79

New electrochemical assay of alkaline phosphatase using ascorbic acid 2-phosphate and its application to enzyme immunoassay  

Microsoft Academic Search

An alternative substrate is described for an enzyme immunoassay with electrochemical detection. Alkaline phosphatase ?ALP) activity is determined by using ascorbic acid 2-phosphate ?AsA-P) as substrate. ALP-generated-AsA is detected amperometrically at a glassy carbon electrode in a flow injection system at +400mV. The optimum assay conditions ?pH, incubation time and concentration of reagent) are examined for the ALP assay. The

Amane Kokado; Hidetoshi Arakawa; Masako Maeda

2000-01-01

80

Comparison of new immunohistochemical assay for oestrogen receptor in paraffin wax embedded breast carcinoma tissue with quantitative enzyme immunoassay  

Microsoft Academic Search

AIM--To validate the use of a new mouse monoclonal antibody (1D5) directed against the N-terminal domain (A\\/B region) of the oestrogen receptor in an immunohistochemical assay (ER-IHA) for paraffin wax embedded tissue. METHODS--Breast cancer specimens were surgically obtained from 119 previously untreated patients. For comparison, oestrogen receptor was measured from cytosol fractions using an established oestrogen receptor enzyme immunoassay (ER-EIA)

G Saccani Jotti; S R Johnston; J Salter; S Detre; M Dowsett

1994-01-01

81

Two rapid and simple enzyme immunoassays for human antibodies to Entamoeba histolytica.  

PubMed

Two rapid and simple enzyme immunoassays (EIA) for antibodies to E. histolytica the protozoa causing ambiasis, are described. In the rapid dot EIA, a qualitative procedure, antigens were dried as a small dot (3 mm in diameter) on a thin white opaque polystyrene strip and serum samples were assayed undiluted. The assay required 3 incubation periods, 1 to 3 minutes each, and was completed in 9 minutes, with a positive reaction revealed as a blue color (precipitate) on the antigen dot and negative as colorless. The developed color is stable for permanent record. In the Microwell EIA, a quantitative procedure, antigens were dried in the Microwells. The assay also consisted 3 incubation periods of 15 minutes each, and was completed in 50 minutes. The results in absorbance values were normalized to EIA units (EU). Both tests had good reproducibility, sensitivity and specificity; and highly correlated with 3 other serologic tests. Their reagents can be stored for more than a year. Both tests could be suitable for small and physicians' office laboratories, especially in developing countries. PMID:2558127

Lin, T M; Schubert, C M; Kiefer, D J; Troy, S; Cort, R; Giegel, J L; Lin, J H; Neill, L

1989-01-01

82

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

PubMed

Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for serum/plasma (IMMULITE and SimulTRAC-SNB) for B12 analysis in human milk. B12-recovery rates (United States Environmental Protection Agency, 2007) were determined to be 78.9 ± 9.1% with IMMULITE and 225 ± 108% (range 116-553%) using SimulTRAC-SNB, most likely due to the presence of excess HC. HC-interferences were not observed with the IMMULITE assay, rendering previously reported mandatory HC-removal (Lildballe et al., 2009) unnecessary. Linearity continued at low B12-concentrations (24-193 pM; r(2)>0.985). Milk B12 concentrations from Bangladeshi women (72-959 pM) were significantly lower than those from California (154-933 pM; p<0.0001) showing IMMULITE's robustness against the complex milk matrix and its ability to measure low milk B12 concentrations. PMID:24491700

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

2014-06-15

83

Enzyme immunoassay for a urinary metabolite of 4-hydroxynonenal as a marker of lipid peroxidation.  

PubMed

Free radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively. PMID:16337879

Guéraud, Françoise; Peiro, Géraldine; Bernard, Hervé; Alary, Jacques; Créminon, Christophe; Debrauwer, Laurent; Rathahao, Estelle; Drumare, Marie-Françoise; Canlet, Cécile; Wal, Jean-Michel; Bories, Georges

2006-01-01

84

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

PubMed

In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL(-1) to 1600 pg mL(-1), and the measured low limit of detection (LOD) was 3.2 pg mL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future. PMID:24016577

Zhang, Chunmei; Liu, Zhijia; Li, Yongming; Li, Qi; Song, Chaojun; Xu, Zhuwei; Zhang, Yun; Zhang, Yusi; Ma, Ying; Sun, Yuanjie; Chen, Lihua; Fang, Liang; Yang, Angang; Yang, Kun; Jin, Boquan

2013-09-24

85

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

PubMed Central

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day. PMID:10747112

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

86

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

87

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

88

Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis.  

PubMed Central

We evaluated a commercially available enzyme-linked immunoassay (ELISA) from LMD Laboratories, Inc., Carlsbad, Calif., for the detection of antibodies in serum to the cysticercus of Taenia solium. The ELISA was performed on 308 serum samples; 198 from a pool of healthy individuals, 42 from patients who had antibodies against a variety of parasites other than T. solium, and 68 from patients suspected of having cysticercosis. All of these 68 specimens were tested both by the ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot assay [EITB]) developed at the Parasitic Serology Laboratory of the Centers for Disease Control and Prevention. Twenty-seven of the 68 serum samples from patients suspected of having cysticercosis were positive by both EITB and ELISA, while 31 were negative by both assays. ELISA results for three and two samples were considered false positive and false negative, respectively, when compared with the results of EITB. Results for an additional five samples were considered equivocal but were technically positive because their optical density readings were slightly above the cutoff value. Three of the 198 serum samples from the bank of serum samples from healthy individuals were also false positive by ELISA (the EITB result for the samples was negative). Six other serum samples from healthy individuals which had equivocal results and the five serum samples from patients with equivocal results were EITB negative. Serum samples containing antibodies against Echinococcus spp. frequently cross-reacted with the cysticercus ELISA antigen (13 of 16 specimens), but serum samples with antibodies against other parasites did not (2 of 26 specimens); all of these serum samples were EITB negative. The commercially available ELISA that we describe is a simple and rapid test. Considering all 308 specimens, the ELISA had a specificity of 93% (when samples with equivocal results were considered negative) or 89% (when samples with equivocal results were considered positive); the sensitivity was 93%. PMID:8586686

Sloan, L; Schneider, S; Rosenblatt, J

1995-01-01

89

Potential Impact of Different Cytomegalovirus (CMV) IgM Assays on an Algorithm Requiring IgM Reactivity as a Criterion for Measuring CMV IgG Avidity  

PubMed Central

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

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

2014-01-01

90

Heterologous enzyme immunoassay for the determination of free indole-3-acetic acid (IAA) using antibodies against ring-linked IAA.  

PubMed

A solid phase indirect enzyme immunoassay method for the plant growth substance indole-3-acetic acid (IAA) using polyclonal antibodies raised to IAA linked to rabbit serum albumin (RSA) is described. The sensitivity for IAA increased by more than three orders of magnitude as the number of IAA ligands on the coating antigen decreased. Further improvements in assay sensitivity were limited by the high affinity of the antibodies for the bridge group in the IAA conjugate. Substitution of the IAA in the coating antigen by either indole-3-propionic acid or indole-3-lactic acid reduced antibody recognition of the bridge group. The resulting heterologous assay compares favourably with existing homologous immunoassays for IAA in terms of sensitivity and specificity. PMID:1995713

Manning, K

1991-01-24

91

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

92

Biotin-avidin amplified enzyme-linked immunosorbent assay (ELISA) for the measurement of canine serum IgA, IgG and IgM.  

PubMed

An amplified capture enzyme-linked immunosorbent assay (ELISA) has been developed by the use of the biotin-avidin detection system, for the measurement of canine plasma immunoglobulins (Ig) A, G and M. Test responses of dilutions of both the Ig standards and test plasma samples were consistently linear (r > 0.987) for the three Ig classes. The within-assay variation was 3.53 per cent for IgG, 5.84 per cent for IgM and 6.34 per cent for IgA. The analytical recoveries were 95 per cent for IgA, 97 per cent for IgG and 98 per cent for IgM. The lower detection limits of the assay were 38.4 ng ml-1 for IgG, 20.3 ng ml-1 for IgM and 41.2 ng ml-1 for IgA. The results indicate that this ELISA has a much higher sensitivity than the single radial immunodiffusion assay or the non-amplified ELISA for measurements of canine Igs, but has a comparable specificity and precision. PMID:8685529

Ginel, P J; Margarito, J M; Molleda, J M; López, R; Novales, M; Bernadina, W E

1996-03-01

93

Development of a simple, rapid sandwich enzyme immunoassay for the measurement of serum rat LH.  

PubMed

The present study describes the development and validation of a rapid, sensitive, specific and precise enzyme immunoassay (EIA) sandwich suitable for measuring luteinizing hormone (LH) in rat serum. Ninety-six well polystyrene microtiter plates were coated with 100 microliters (250 ng/ml) of a well-characterized monoclonal antibody (518B7, Roser, UC Davis) generated against bovine LH. A polyclonal antiserum raised in rabbits against ovine FSH (G4-215B, Papkoff) was conjugated to sodium periodate-activated horseradish peroxidase (HRP), and used as the second antibody of the sandwich assay. This anti-ovine FSH antiserum cross-reacted more than 200% with rat LH. Standards (r-LH-RP-3, NIADDK, range 0 pg/well to 2.5 ng/well or 100 microliters) diluted in a 3(N-Morpholino) propane sulfonic acid (MOPS) buffer, or serum, were incubated with the solid phase antibody for 2 hours. Plates were washed and the anti-oFSH:HRP (100 microliters) in MOPS buffer was added and incubated a further 2 hours before a second wash and the addition of the substrate (TMB, 3,3',5,5'-tetramethylbenzidine dihydrochloride and H2O2). The least detectable concentration of LH was 16.1 +/- 1.42 pg/ml. The recovery of known concentrations of LH added to several samples was 93.5 +/- 1.70%. Mean intra-assay and inter-assay coefficients of variation (%) were less than 10% (n = 20). The anti-FSH:HRP showed less than 8.0% cross reactivity with rFSH in this LH EIA system. The correlation coefficient (r) of samples analyzed by EIA in parallel with RIA was r = 0.90 (p < 0.001, n = 26). Results showed levels between 105.21 and 633.87 pg/ml. This new LH EIA sandwich offers a stable, rapid, and improved EIA system for the measurement of serum LH concentrations of this species over previously reported methods. PMID:8870107

Illera, J C; Munro, C J; Silvan, G; BonDurant, R H; Illera, M

1996-06-01

94

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan  

Microsoft Academic Search

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and

Shi-Yuan Sheu; Yi-Chih Lei; Yung-Te Tai; Tong-Hsuan Chang; Tzong-Fu Kuo

2009-01-01

95

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...

96

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

97

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

98

Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein.  

PubMed

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

99

Magnetic Affinity Enzyme-Linked Immunoassay for Diagnosis of Schistosomiasis Japonicum in Persons with Low-Intensity Infection  

PubMed Central

Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections. PMID:22869635

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

2012-01-01

100

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

101

Enzyme Inhibitor Screening Using a Homogeneous Proximity-Based Immunoassay for Estradiol  

Microsoft Academic Search

The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near-infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved

Leena Kokko; Nina Johansson; Timo Lövgren; Tero Soukka

2005-01-01

102

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

Microsoft Academic Search

Immunoassays in pesticide residue analysis significantly profit from the monoclonal antibody (mAb) technology because a sufficient supply of standardized antibodies can be provided. For the production of atrazine-specific mAbs hybridoma cells were produced by fusion of mouse myeloma cells and spleen cells from mice which were immunized with 4-chloro-6-ethylamino- 1,3,5-triazine-2-(6-aminohexanoic acid) coupled to keyhole limped hemocyanin. After screening with a

Bertold Hock; Thomas Giersch; Karl Kramer

1993-01-01

103

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

104

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

105

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...

106

Rises in antibody to human herpesvirus 6 detected by enzyme immunoassay in transplant recipients with primary cytomegalovirus infection.  

PubMed Central

Immunoglobulin G to human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) in sera from solid organ recipients was measured by an enzyme-linked immunoassay (ELISA) before and after transplant. The HHV-6 ELISA was developed from glycine extracts of HHV-6-infected and uninfected HSB-2 cells. At a serum dilution of 1:500, 80 (91%) of 88 recipients were seropositive for HHV-6 before transplant, while only 14 (16%) were seropositive for CMV. Posttransplant HHV-6 serologic rises were observed in 38 (43%) recipients; rises in 25 of these recipients were associated with primary CMV infection. Titration of sera revealed much higher HHV-6 titer rises among those with primary CMV infection than among those with CMV reactivation or with no CMV infection. Elevated HHV-6 antibody titers persisted for up to 2 years after primary CMV infection. No correlation was noted between CMV and HHV-6 antibody titers in individual serum samples. PMID:2161867

Chou, S W; Scott, K M

1990-01-01

107

Comparison of eight commercial enzyme immunoassays for the detection of Clostridium difficile from stool samples and effect of strain type.  

PubMed

We compared the performance of 8 Clostridium difficile enzyme immunoassays to cell cytotoxicity neutralization assay and toxigenic culture. The effect of strain type on assay performance was also examined. There were a total of 71 (14.4%) samples in which C. difficile was recovered; 58 (81.7%) of 71 were toxigenic. Compared to a composite reference standard of either C. difficile cytotoxin assay positive or toxigenic C. difficile culture positive, the sensitivities of these assays varied from 31.7% to 55.2%, while the specificities were excellent, ranging from 98.1% to 100%. Among the 57 C. difficile isolates, 30 (51.7%) were of the NAP1 genotype. Stool samples positive for the C. difficile NAP1 strain had a higher positivity rate for the toxin assays. PMID:22424900

René, Pierre; Frenette, Charles P; Schiller, Ian; Dendukuri, Nandini; Brassard, Paul; Fenn, Susan; Loo, Vivian G

2012-05-01

108

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

109

Comparison of a New Neuraminidase Detection Assay with an Enzyme Immunoassay, Immunofluorescence, and Culture for Rapid Detection of Influenza A and B Viruses in Nasal Wash Specimens  

Microsoft Academic Search

The performance of a new, rapid, easy-to-perform assay based on neuraminidase enzyme activity for detection of influenza virus types A and B was compared to detection by culture, indirect immunofluorescence, and enzyme immunoassay in 479 nasal wash specimens from children with respiratory infections. Compared to isolation of influenza virus by culture, the neuraminidase assay had a sensitivity of 70.1%, specificity

DANIEL E. NOYOLA; BRUCE CLARK; FREDERICK T. O'DONNELL; ROBERT L. ATMAR; JEWEL GREER; GAIL J. DEMMLER

2000-01-01

110

Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load.  

PubMed

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10(3) U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 x 10(2) U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients. PMID:11825954

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

2002-02-01

111

Enzyme-linked immunoassay for detection of Listeria monocytogenes in dairy products, seafoods, and meats: collaborative study.  

PubMed

A collaborative study was conducted to evaluate Listeria-Tek, an enzyme-linked immunosorbent assay (ELISA) for detection of Listeria monocytogenes and other Listeria spp. in foods. The present ELISA method was compared to the U.S. Food and Drug Administration culture method for detection of L. monocytogenes in dairy products and seafoods and to the U.S. Department of Agriculture Food Safety and Inspection Service method for detection of L. monocytogenes in meats. Replicate samples of 6 food types (frankfurters, roast beef, Brie cheese, 2% milk, raw shrimp, and crab meat) inoculated with L. monocytogenes and uninoculated control samples were analyzed by the collaborators. L. monocytogenes was identified in 593 samples by the ELISA method and in 574 samples using culture procedures. Identical results were obtained for 506 positive samples and 419 negative samples using the ELISA and culture methods for an overall agreement rate of 85.6%. The enzyme-linked immunoassay for detection of L. monocytogenes in dairy, seafood, and meat products has been adopted first action by AOAC INTERNATIONAL. PMID:7819756

Curiale, M S; Lepper, W; Robison, B

1994-01-01

112

Predicting behavior of an enzyme-linked immunoassay model by using commercially available neural network software.  

PubMed

Setting up new immunoassays can be a laborious and expensive task. A relatively new form of multivariate analysis known as neural networks can be applied to this problem with potential savings in reagents and technician time. Neural network software programs for personal computers are now available. We applied one such software package (Brainmaker) to a model ELISA system for measuring human serum albumin. Random combinations of four variable ELISA conditions (antigen concentration, primary and secondary antibody titers, and time for chromagen development) were used to train a three-layered feed-forward network. The trained network was then used to predict measured absorbances as a function of the four input variables in separate cross-validation sets. The network adequately predicted the effect of the input variables on the absorbance produced. With use of such methods, optimal conditions for the linear dependence of absorbance on antigen concentration can be evaluated on the computer rather than in the laboratory, with subsequent savings of time and money. PMID:8252719

Vertosick, F T; Rehn, T

1993-12-01

113

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

PubMed

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months. PMID:21242086

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

2011-03-15

114

A Highly Sensitive Sandwich Enzyme Immunoassay of Human Growth Hormone in Serum Using Affinity-Purified Anti-Human Growth Hormone Fab?Horseradish Peroxidase Conjugate  

Microsoft Academic Search

A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were

Seiichi Hashida; Koji Nakagawa; Shinji Yoshitake; Masayoshi Imagawa; Eiji Ishikawa; Yuichi Endo; Sachiya Ohtaki; Yutaka Ichioka; Katsuyuki Nakajima

1983-01-01

115

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

116

Stool Diagnosis ofGiardiasis Using a Commercially Available EnzymeImmunoassay ToDetect Giardia-Specific Antigen 65(GSA65)  

Microsoft Academic Search

A commercially available enzyme immunoassay forthediagnosis ofgiardiasis was evaluated ina clinical trial. TheProSpecT\\/Giardia diagnostic test(Alexon, Inc., Mountain View,Calif.) was compared withthe standard ova andparasite (O&P)microscopic examination. Additionally, several widely usedstool fixatives andacommonly usedtransport mediumwereassessed forcompatibility withtheimmunoassay. A total of325 stool specimens were collected andusedtoevaluate assayperformance. Ofthose, 93specimens were collected fromsymptomatic Giardia O&P-positive patients and232specimens were randomly collected frompatients as partofa

JOHN D. ROSOFF; CYNTHIA A. SANDERS; SEEMA S. SONNAD; PAUL R. DE LAY; W. KEITH HADLEY; FRANK F. VINCENZI; DAVID M. YAJKO; PETER D. O'HANLEY

117

Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4\\/heparin enzyme-immunoassay  

Microsoft Academic Search

BackgroundIgG-specific anti-PF4\\/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain.

Karina Althaus; Ulrike Strobel; Theodore E. Warkentin; Andreas Greinacher

2011-01-01

118

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

119

Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk  

Technology Transfer Automated Retrieval System (TEKTRAN)

BACKGROUND Few accurate data exist on the concentration of vitamin B12 in human milk. Binding of the vitamin to haptocorrin (HC) can interfere with the assay if not removed by pretreatment, and very low values can occur in women with poor B12 status. This study evaluated two competitive enzyme bind...

120

Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas).  

PubMed

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM. PMID:16519293

Brodeur, Julie C; Woodburn, Kent B; Zhang, Fagen; Bartels, Michael J; Klecka, Gary M

2006-02-01

121

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins.  

PubMed Central

A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children. Images PMID:3531231

Belmaaza, A; Hamel, J; Mousseau, S; Montplaisir, S; Brodeur, B R

1986-01-01

122

Screening test for rheumatic diseases: a combined enzyme immunoassay of rheumatoid factors and antibodies to DNA and extractable nuclear antigens.  

PubMed Central

Three hundred and one sera from patients with rheumatic and other diseases were investigated using a simple enzyme immunoassay for screening of rheumatoid factors and antinuclear antibodies. The assay had a sensitivity of 77% for systemic lupus erythematosus, 90% for the primary sicca syndrome, and 89% for rheumatoid arthritis. Only 13% of sera from patients with chronic non-rheumatic diseases were positive. The test was further evaluated in a group of patients with suspected rheumatic disease who were followed up for six to 12 months. The test was positive in 16 of 17 sera from patients with connective tissue diseases but in only seven of 36 sera (19%) from patients with non-inflammatory joint diseases. None of the four patients with reactive arthritis was positive by this test. The sensitivity of the assay was comparable with that of the agglutination and immunofluorescence tests for rheumatoid factors and antinuclear factors. For the screening of rheumatoid factor and antinuclear antibodies this kind of test panel offers a simple alternative to the conventional tests for small clinical laboratories and for those in which the autoantibody tests could be automated, as the assay can be performed in one working day and only one dilution of serum is needed to obtain a quantitative result. Images Figure PMID:3323254

Kurki, P; Gripenberg, M; Partanen, P; Helve, T

1987-01-01

123

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

124

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

PubMed Central

Monoclonal antibodies specific for the envelope (E1), peplomer (E2), and nucleocapsid (N) polypeptides of feline infectious peritonitis virus (FIPV) were used in rapid, competitive enzyme-linked immunosorbent assays (ELISA) to study the humoral immune response of cats to FIPV infection. Results from the competitive ELISAs were correlated with those from immunofluorescent antibody assays (IFAs) on 203 samples obtained from 64 individual cats. The IFA results correlated best with those obtained with the anti-E1 specific competitive ELISA (85.7%). In contrast, anti-N and anti-E2 competitive ELISA results correlated with IFA results only 65.5 and 2.4% of the time, respectively. The results of the anti-E1 specific competitive ELISA were not influenced by the total immunoglobulin concentration or the possible presence of free viral antigens in the serum. These results suggest that a competitive ELISA involving the use of enzyme-conjugated monoclonal antibody to the E1 glycoprotein of FIPV is a simple and rapid replacement for the more cumbersome IFA. Images PMID:2995437

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

1985-01-01

125

Magnetic particle-based enzyme assays and immunoassays for microcystins: from colorimetric to electrochemical detection.  

PubMed

In this work, magnetic particles (MPs) are used as supports for the immobilization of biorecognition molecules for the detection of microcystins (MCs). In one approach, a recombinant protein phosphatase 1 (PP1) has been conjugated to MPs via coordination chemistry, and MC-LR detection has been based on the inhibition of the enzyme activity. In the other approach, a monoclonal antibody (mAb) against MC-LR has been conjugated to protein G-coated MPs, and a direct competitive enzyme-linked immunoparticle assay (ELIPA) has been then performed. Conjugation of biomolecules to MPs has been first checked, and after optimization, MC detection has been performed. The colorimetric PPIA with PP1-MP and the best ELIPA strategy have provided limits of detection (LOD) of 7.4 and 3.9 ?g/L of MC-LR, respectively. The electrochemical ELIPA has decreased the LOD to 0.4 ?g/L, value below the guideline recommended by the World Health Organisation (WHO). The approaches have been applied to the analysis of a cyanobacterial culture and a natural bloom, and MC equivalent contents have been compared to those obtained by conventional assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results have demonstrated the viability of the use of MPs as biomolecule immobilization supports in biotechnological tools for MCs monitoring. PMID:23214443

Reverté, Laia; Garibo, Diana; Flores, Cintia; Diogène, Jorge; Caixach, Josep; Campàs, Mònica

2013-01-01

126

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

127

Labeled anti-hapten antibodies and their use as a universal reagent for solid phase radio and/or enzyme-immunoassays  

SciTech Connect

A process for detecting the presence of an antigen in a specimen is described, which process comprises: contacting said specimen with a substrate coated with antibodies of said antigen, incubating the contacted substrate and washing the substrate; contacting the washed material of step with a hapten conjugated antibody against said antigen, incubating the so-contacted material and washing the so-incubated material; contacting the washed material of step with a radioactive material labeled or enzyme containing anti-hapten antibody, incubating the so-contacted material and washing the same; and effecting radioimmunoassay if said antibody is radioactive or enzyme labeled immunoassay if said antibody contains an enzyme moiety. Quantitative determination of the antigen in the specimen is effected by comparing the counts of the radioimmunoassay or the concentration of enzyme against a standard as by photocolormetric methods.

Neurath, A. R.; Strick, N.

1985-01-22

128

Rapid detection of parainfluenza virus type 3 RNA in respiratory specimens: use of reverse transcription-PCR-enzyme immunoassay.  

PubMed Central

Parainfluenza virus type 3 (PIV-3), an important lower respiratory tract pathogen in young children and immunocompromised individuals, may be underdiagnosed because of the insensitivity of available culturing systems and delay in identification of virus in cell culture. We developed a reverse transcription-PCR-enzyme immunoassay (RT-PCR-EIA) for PIV-3, using primers specific for a highly conserved region of the hemagglutinin-neuraminidase gene. Testing of nasal washes spiked with PIV-3 or other respiratory viruses showed that this assay detected seven strains of PIV-3 but not other respiratory viruses. Of 103 respiratory tract samples obtained from children experimentally infected with a liver PIV-3 vaccine or naturally infected with wild-type PIV-3, 51 were positive by culture and 48 were positive by RT-PCR-EIA. Eleven of the culture-positive samples were negative by RT-PCR-EIA; however, none of these grew virus upon reinoculation into cell culture, indicating that virus was lost or was present at a very low titer. Eight of the culture-negative samples were positive by RT-PCR-EIA: two were obtained from a subject who was culture negative but had a serologic response to PIV-3, four were obtained 7 to 9 days after the first positive culture, and two were obtained 1 day prior to the first positive culture. Thus, this RT-PCR-EIA for PIV-3 is sensitive and specific and can detect viral RNA in samples from which virus cannot be cultivated. This assay could be used for diagnosis late in the course of PIV-3 infection and for accurate detection of disease outbreaks. Images PMID:8150961

Karron, R A; Froehlich, J L; Bobo, L; Belshe, R B; Yolken, R H

1994-01-01

129

Enzyme immunoaffinity chromatography--a rapid semi-quantitative immunoassay technique for screening the presence of isoproturon in water samples.  

PubMed

Immunochromatography devices based on the principles of affinity chromatography and enzyme immunoassay have been developed to illustrate the possibility of providing extra-laboratory 'in the field' tests for pesticide monitoring. Isoproturon was chosen in this study as an example though other pesticides could have been used provided a suitable antiserum existed. The test system was prepared by immobilising isoproturon antibodies to porous silica which were then packed into disposable columns (immunoaffinity columns). The addition of chromagen-substrate 3,3',5,5'-tetramethylbenzidine into the immunoaffinity columns, after the application of a mixture containing an equal volume of isoproturon samples or isoproturon standard solutions with a fixed concentration of isoproturon labelled with horseradish peroxidase, allowed the development of a colorimetric portable assay capable of screening samples qualitatively for the presence of isoproturon in water. Samples with contamination levels of 0.12 microgram l-1 isoproturon and above were visually identified as positive samples in comparison to the zero standard sample. However, samples with concentrations below this level would be considered as negative (no isoproturon present at this limit of detection). The duration of the sample screening procedure on the column was less than 25 min making the technique highly suitable for a rapid estimate of isoproturon concentrations. Furthermore, the system could estimate isoproturon concentrations in water from various sources with no requirement for sample preparation. Therefore, the technique is ideally suited for monitoring the presence of pesticides in water. Further refinement of the assay could result in a test suitable for use by non-skilled personnel in extra-laboratory locations (e.g., a mobile or field laboratory). PMID:9246817

Katmeh, M F; Godfrey, A J; Stevenson, D; Aherne, G W

1997-05-01

130

Clinical evaluation of a bioluminescent enzyme immunoassay for detecting norovirus in fecal specimens from patients with acute gastroenteritis.  

PubMed

Noroviruses (NoVs), which belong to the family Caliciviridae, are major causative agents of acute gastroenteritis worldwide. Thus, rapid and highly sensitive assays for detecting NoVs are required. Recently, a bioluminescent enzyme immunoassay (BLEIA) for detecting NoVs in fecal specimens was developed. This new assay was evaluated using fecal specimens obtained from acute gastroenteritis patients. Of the 107 specimens that were found to be NoV-positive by RT-PCR or RT-LAMP, 104 specimens produced positive results in the BLEIA (sensitivity: 96.3%). On the other hand, no false-positive results were observed during the testing of 176 NoV-negative specimens containing group A or C rotaviruses, astroviruses, sapoviruses, adenovirus type 41, bocaviruses, or parechoviruses. Furthermore, the BLEIA was able to detect many NoV genotypes in the tested specimens, including three genotypes from genogroup I (genotypes 1, 4, and 8) and ten genotypes belonging to genogroup II (genotypes 1, 2, 3, 4, 5, 6, 12, 13, 16, and 19). By quantifying the number of NoV genome copies in the clinical specimens tested with the BLEIA, its detection limit was estimated to be 10(6) genome copies per gram of stool and below. Furthermore, as the BLEIA can be performed with an automated device and does not involve complicated procedures it can be used to rapidly test many samples. Therefore, the BLEIA is a rapid and highly sensitive method and could be used as a diagnostic tool at hospitals and clinical laboratories that deal with large numbers of clinical specimens from acute gastroenteritis patients or food handlers. J. Med. Virol. 86:1219-1225, 2014. © 2013 Wiley Periodicals, Inc. PMID:24114991

Shigemoto, Naoki; Tanizawa, Yukie; Matsuo, Takeshi; Sakamaki, Nozomi; Ohiro, Yoshiyuki; Takayasu, Susumu; Fukuda, Shinji

2014-07-01

131

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

132

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

133

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

PubMed

Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

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

2014-04-01

134

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

PubMed Central

Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

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

2014-01-01

135

Performance of a conventional enzyme immunoassay for hepatitis C virus core antigen in the early phases of hepatitis C infection.  

PubMed

There are periods within the early phase of hepatitis C virus (HCV) infection in which the anti-HCV antibody test is unable to confirm HCV viremia. To reduce the risk of transmitting HCV through transfusions, we developed a simple and highly sensitive enzyme immunoassay (EIA) which detects the core antigen of HCV (HCVcAg). This assay employed a conventional colorimetric EIA system, and was based on a two-step sandwich assay, using a 96- well microplate. The reproducibility of the results was very high. When the cutoff values were set to 30 fmol of recombinant HCVcAg/L, as determined by the distribution of healthy subject sera (n=223), 99.6% of healthy subject sera and 100% of hepatitis B patient sera (n=50) were negative for HCVcAg. The clinical performance of this EIA was examined using 14 commercially available seroconversion panels. In every panel, HCVcAg could be detected at points preceding the seroconversion of anti-HCV antibodies. The points at which HCVcAg was detected were the same as those at which it was detected by an AMPLICOR HCV Monitor test. The EIA's window period for detecting the HCVcAg in all panels was on average 26 days shorter than that of the anti-HCV antibody test. In three panels where the first sample is negative for HCV RNA, the window period was shortened 50 days by this EIA for HCVcAg. There was a positive correlation between the concentration of HCVcAg and HCV RNA in anti-HCV antibody negative specimens. This assay was simpler to perform than assays based on gene amplification technology for the detection of HCV RNA, and the window period was shortened to that of the AMPLICOR HCV Monitor test. Thus, the EIA for HCVcAg would be useful in screening seroconverting donors and could reduce the residual risk of secondary HCV infections through transfusions. PMID:11294574

Aoyagi, K; Iida, K; Ohue, C; Matsunaga, Y; Tanaka, E; Kiyosawa, K; Yagi, S

2001-01-01

136

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

PubMed Central

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

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

2005-01-01

137

Comparison of sensitivity of enzyme immunoassays for toxin A and B in different C. difficile PCR ribotypes.  

PubMed

Enzyme immunoassays (EIAs) for toxins A and B are the most common assays for the diagnosis of Clostridium difficile infection due to their rapidity and ease of use. However, the sensitivity of different kits varies greatly. The predominant PCR ribotypes of C. difficile vary according to the region or country studied, and it was recently reported that the sensitivity of EIAs can be affected by the strain type. The aim of this study was to assess the sensitivity of EIAs in different PCR ribotypes of C. difficile during a period of five years in a Korean hospital. A total of 969 toxigenic C. difficile isolates were recovered from patients with diarrhea in a hospital from 2006 to 2009 (inclusive), and 2011. Overall sensitivities of Tox A/B Quik Chek (TechLab, Blacksburg, VA) and VIDAS C. difficile A & B (bioMérieux, Marcy l'Etoile, France) were 36.4% and 46.3%, respectively. The sensitivities of TOX A/B Quick Chek and VIDAS Clostridium difficile A & B for the five most common ribotypes were as follows: 56.6% and 71.7% for ribotype AB17 (ribotype 018); 48.6% and 54.3% for ribotype aB (ribotype 017); 25.3% and 36.3% for ribotype AB2 (ribotype 014); 13.0% and 24.2% for ribotype AB3; 66.7% and 0% for ribotype AB1 (ribotype 001), respectively. The sensitivity for the predominant ribotype, AB17, was significantly different from those for aB, AB2, AB1, and AB3 using VIDAS Clostridium difficile A & B (p<0.05). These data suggest that the sensitivity of EIA may be affected by the distribution of ribotypes. PMID:24695472

Lee, Yangsoon; Kim, Myungsook; Kim, Heejung; Lee, Kyungwon

2014-01-01

138

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

PubMed Central

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

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

2014-01-01

139

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

140

Enzyme-linked Fab' fragment based competitive immunoassay for ovalbumin in hot-processed food.  

PubMed

A hen's egg is one of the most common causes of food allergy. The allergen quantitation in hot-processed food is always a difficult task, because the protein in these samples will be denatured, insoluble, and degraded. This article presents a competitive enzyme linked immunosorbent assay (ELISA) for the quantitation of ovalbumin in hot-processed food. Its recovery was improved nearly two times by the assay method as compared with previous sandwich ELISA. The heat and DL-Dithiothreitol treated ovalbumin was used as antigen for monoclonal antibody preparation. A smaller labeled antibody molecule, horseradish peroxidase (HRP) labeled Fab' fragment, was used to replace IgG in ELISA for improving sensitivity and analytical speed of the method. A binding site protection procedure was developed for Fab' fragment labeling with HRP, which prevented damage to the Fab' binding site. The combination and separation steps were efficiently completed in an affinity spin column. Based on the optimized ELISA condition, the IC50 was 1.2 µg/mL and the coefficients of variation were less than 10%. PMID:23859790

Zhang, Shiwei; Lai, Xintian; Yang, Guowu

2013-01-01

141

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

142

The effect of storage conditions on salivary cortisol concentrations using an Enzyme Immunoassay.  

PubMed

Abstract 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

143

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

PubMed

Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in-house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n=72), (B) samples from known HEV-negative pigs (negative controls, n=62) and (C) samples from pigs of unknown HEV infection status (n=182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa=0.92). PMID:23773809

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

2013-11-01

144

Determination of the concentrations of the steroids estradiol, progesterone and testosterone in bovine sera: comparison of commercial dissociation enhanced lanthanide fluorescence immunoassay kits with conventional radio and enzyme immunoassays.  

PubMed

The performance of three conventional enzyme and radioimmunoassays routinely used to detect residues of anabolic steroids in cattle sera were compared with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) kits designed for the hospital market. Slight modifications to the kit reagents were required for the analysis of bovine sera. Owing to the large sample volumes used in conventional assays, detection limits were generally better than those obtained with DELFIA kits, however, assay reproducibility was enhanced using the DELFIA technology. Comparison of sera obtained from cattle implanted with anabolic steroids revealed a good correlation between alternate methods (r2 from 0.91 to 0.97). The DELFIA kits offer a faster method for measuring estradiol, progesterone and testosterone with adequate sensitivity and in a safer environment than that encountered using radioimmunoassays. PMID:7604958

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

1995-06-01

145

Anti-idiotype scFv-enzyme fusion proteins: a clonable analyte-mimicking probe for standardized immunoassays targeting small biomarkers.  

PubMed

Most immunoassays use probes that convert concentrations of analytes into signal intensity. To prepare the probes, analytes are usually linked to a reporter protein (e.g., enzymes) with the aid of chemical reagents. However, these conventional methods yield a mixture of heterogeneous products and consequently reduce assay performance. "Clonable" homogeneous probes, i.e., recombinant molecules in which a target protein is genetically fused to a reporter with a defined coupling ratio, are now available for analyzing protein biomarker concentrations. Here, we have expanded this strategy to measure small biomarkers (haptens) by genetically fusing proteinaceous molecules that mimic target haptens with enzymes. 11-Deoxycortisol (11-DC) was chosen as the model hapten, and the ?-type anti-idiotype antibodies (?Id-Abs) that recognize the paratope of anti-hapten antibodies were used as the target hapten mimic. The V(H) and V(L) genes of a ?Id-Ab, targeting a mouse anti-11-DC antibody (CET-M8), were assembled to encode a single-chain Fv fragment (?Id-scFv), which was then fused with a gene encoding a variant of alkaline phosphatase. The product, ?Id-scFv-ALP' protein, had satisfactory enzyme activity and bound to CET-M8 in a competitive manner with 11-DC. A colorimetric enzyme-linked immunosorbent assay (ELISA) for 11-DC, based on the competitive reaction between the analyte and ?Id-scFv-ALP' against immobilized CET-M8, was found to be sensitive (limit of detection = 22 pg/assay) and specific (cross-reactivity with cortisol, 0.24%) for clinical use and could be used to determine serum 11-DC levels after a simple solvent extraction. The anti-idiotype scFv-enzyme fusion proteins proposed here can be prepared reproducibly as homogeneous products with a 1:1 coupling ratio and would facilitate standardization of immunoassays for small biomarkers. PMID:24256209

Oyama, Hiroyuki; Tanaka, Eri; Kawanaka, Tomoyo; Morita, Izumi; Niwa, Toshifumi; Kobayashi, Norihiro

2013-12-01

146

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology.  

PubMed

To develop improved reagents for mumps serology a high-level yeast expression system was employed to produce recombinant mumps nucleoprotein (rNP). The rNP was purified by CsCl gradient centrifugation and yielded approximately 15 mg/l of yeast culture. Electron microscopy of the rNP revealed characteristic herring-bone structures. The electrophoretic mobility of rNP in yeast cells was similar to native NP in SDS-PAGE. Monoclonal antibodies to rNP reacted with native mumps virus nucleoprotein by immunofluorescence assay. A monoclonal antibody to native mumps virus NP reacted with rNP by Western blot assay. The rNP was investigated as antigen in an IgM capture enzyme immunoassay (EIA) using a horseradish peroxidase conjugate of monoclonal antibody to the rNP. Eighteen sera previously found to be positive by IgM capture radioimmunoassay (MACRIA) and 30 sera that were mumps IgM negative by MACRIA were tested by mumps IgM capture EIA. The results for the two test were concordant. In addition, 26 rheumatoid factor positive sera and 35 sera that were IgM positive for measles, rubella or parvovirus B19 were tested. Fifty-nine sera were negative by mumps IgM capture EIA but two sera collected from two infants 3 and 6 weeks after mumps, measles and rubella vaccination were positive. Mumps MACRIA confirmed these results. Compared to MACRIA the overall sensitivity was 100% (20/20) and specificity was 96.8% (30/31). The yeast expressed rNP was highly immunogenic and suitable for use in IgM capture EIA for the diagnosis of mumps. PMID:11748668

Samuel, Dhanraj; Sasnauskas, Kestutis; Jin, Li; Beard, Stuart; Zvirbliene, Aurelija; Gedvilaite, Alma; Cohen, Bernard

2002-01-01

147

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

Microsoft Academic Search

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

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

2004-01-01

148

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

PubMed

We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10-methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC-CL method and 6.2 × 10(-20) mol ?-D-galactosidase (?-gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC-CL method. This highly sensitive CL ?-gal assay was applied to an EIA for thyroid-stimulating hormone (TSH) using ?-gal as a label enzyme; 0.02-100.0 ?U/mL TSH in human serum could be assayed directly and with high reproducibility. PMID:23832789

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

2014-06-01

149

Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies.  

PubMed

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis. PMID:11063489

Groen, J; Koraka, P; Velzing, J; Copra, C; Osterhaus, A D

2000-11-01

150

Saliva as a source of anti-Toxoplasma gondii IgG for enzyme immunoassay in human samples.  

PubMed

Toxoplasmosis, a highly prevalent disease, is mainly diagnosed by serology. Incidence studies could be feasible in children, but ethical concerns restrict blood sampling in this group. Saliva contains small amounts of crevicular fluid IgG. Dot-ELISA and a protein A IgG capture immunoassay were standardized for anti-Toxoplasma gondii IgG in paired saliva and serum samples from 20 adult volunteers. A frequency of toxoplasmosis of 19% (95% CI 12-28) was found in 100 saliva samples from university graduates using both assays. Toxoplasmosis immunoassays using saliva IgG are a promising tool for the investigation of the epidemiology of this disease in children and other vulnerable groups. PMID:23848317

Sampaio, B F C; Macre, M S; Meireles, L R; Andrade, H F

2014-01-01

151

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

152

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

PubMed

A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of beta-lactam antibiotics in raw, commingled milk (Fluorophos BetaScreen E. U. test) was evaluated. A dose-response profile for penicillin G was constructed by analysis of spiked milk samples. The limit of detection, defined as the concentration of penicillin G that resulted in 95% of the samples being evaluated as positive, was 1.8 micrograms/kg. The repeatability of the assay was very high both within and between the three participating milk quality testing laboratories. In total 5,061 randomly selected tanker milk samples were analyzed with the BetaScreen test and compared with the Delvotest SP. The agreement between the two tests was 99.7%. Probably due to a higher sensitivity to penicillin G, the BetaScreen test detected almost twice as many suspect positive tanker milk samples (0.45%) as the Delvotest SP (0.26%). PMID:9678160

Sternesjö, A; Johnsson, G

1998-07-01

153

Quantification of lipoprotein lipase (LPL) by dissociation-enhanced lanthanide fluorescence immunoassay. Comparison of immunoreactivity of LPL mass and enzyme activity of LPL.  

PubMed

Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and in very low density lipoproteins. In this study, a new sensitive enzyme immunoassay, the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), for the quantification of immunoreactive LPL mass in biological specimens was developed. In the indirect sandwich DELFIA assay polyclonal anti-human or anti-bovine LPL IgGs were used as capture antibodies, monoclonal antibody (mAb) 5D2 and Eu(3+)-labelled goat anti-mouse IgG were used as detection antibodies. In the direct sandwich DELFIA assay, mAb 5D2 was used as capture and Eu(3+)-labelled mAb 5D2 as detection antibodies. Both purified bovine and human LPL proteins served as standards in the indirect and the direct DELFIA assay. Standard curves were linear between 0.1 and 1000 ng LPL/ml, assuring the sensitivity of the DELFIAs within this range. Mean values for immunoreactive LPL mass in normal individuals were found to be 40.3 +/- 14.4 ng/ml preheparin plasma and 334.1 +/- 71 ng/ml postheparin plasma. In patients affected with type I hyperlipoproteinemia 82.4 +/- 29.3 ng/ml (postheparin plasma) were determined. Coefficients of inter- and intra-assay variation were 4.3% and 6.2% on average. The correlation coefficient between the indirect and the direct DELFIA technique was 0.9694. The correlation coefficient between immunoreactive LPL mass (estimated by DELFIA) and LPL activity (estimated by the LPL activity assay) was 0.9345. Our data are consistent with the concept that LPL is active as a dimer. Dissociation of the LPL dimer into monomers is tightly coupled to both loss of immunoreactivity and enzyme activity of LPL. PMID:8699004

Wicher, I; Sattler, W; Ibovnik, A; Kostner, G M; Zechner, R; Malle, E

1996-06-10

154

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 Central

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

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

2012-01-01

155

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

156

Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay  

PubMed Central

Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (? = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ?50% showed very good inter-rater agreement (? = 0.968) with V-NA titers of ?1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ?1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections. PMID:10074525

von Messling, Veronika; Harder, Timm C.; Moennig, Volker; Rautenberg, Peter; Nolte, Ingo; Haas, Ludwig

1999-01-01

157

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

158

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

PubMed

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. PMID:25622607

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

2015-02-15

159

Detection of ferritin in human serum with a MAP-H(2)O(2)-HRP voltammetric enzyme-linked immunoassay system.  

PubMed

A voltammetric enzyme-linked immunoassay based on new system of m-aminophenol (MAP)-H(2)O(2)-horseradish peroxidase (HRP) has firstly been developed and used for the detection of HRP, labelled HRP and ferritin in human serum. HRP or labelled HRP catalyzes the oxidation reaction of MAP with H(2)O(2), the product of which produces a sensitive voltammetric peak at potential of -0.46 V (vs. SCE) in Britton-Robinson (BR) buffer solution. By using this voltammetric peak, HRP can be measured with a detection limit of 9.5x10(-1) mU/l and a linear range of 2.5-2.5x10(2) mU/l. The detection limit to ferritin is 0.25 ng/ml and the linear range 0.25-320 ng/ml. The processes of the electro-reduction of the product of the enzyme-catalyzed reaction have been investigated in detail. PMID:18967699

Zhang, S; Jiao, K; Chen, H; Wang, M

1999-08-23

160

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

PubMed Central

A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (Phospho-AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture Phospho-AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated proteins. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of Phospho-AChE in human plasma with a detection limit of 0.020 nM. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures. PMID:19135350

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

2009-01-01

161

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

SciTech Connect

A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.

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

2009-03-01

162

Enzyme-linked immuno-filtration assay (ELIFA) for the detection of IgG, IgM, IgA and IgE antibodies against Aspergillus fumigatus.  

PubMed

The enzyme-linked immuno-filtration assay (ELIFA) permits detection of serological precipitating systems preformed by immunoelectro-diffusion on cellulose acetate strips and simultaneous characterization of immunoglobulins G, M, A and E specific for antigens of Aspergillus fumigatus. We selected 36 sera from 9 patients who were followed up regularly and who suffered from aspergilloma (5 cases), allergic bronchopulmonary aspergillosis (2 cases), Aspergillus bronchitis and invasive aspergillosis (one case each). All of them possessed an IgG-reactive band with chymotrypsin activity. Four different IgE bands were found by ELIFA; one of them was common to all the patients who had anti-A. fumigatus IgE (7 cases out of 9). The IgA and IgM antibodies found in 7 cases out of 9 were both recognized by the same antigenic component but these fractions were distinct from those reacting with the IgE. PMID:3110398

Pinon, J M; Thoannes, H; Poirriez, J; Boulant, J; Lepan, H

1987-04-01

163

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

164

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

Microsoft Academic Search

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

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

2001-01-01

165

Detection of Aspergillus Galactomannan: Comparison of an Enzyme-Linked Immunoassay and a Europium-Linked Time-Resolved Fluoroimmunoassay  

PubMed Central

With a view to improving the sensitivity of serological detection of Aspergillus galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay. PMID:9738075

Paugam, A.; Sarfati, J.; Romieu, R.; Viguier, M.; Dupouy-Camet, J.; Latgé, J. P.

1998-01-01

166

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

167

Comparison of enzyme immunoassay, PCR, and type-specific cDNA probe techniques for identification of group A rotavirus gene 4 types (P types).  

PubMed Central

This study was designed to evaluate three techniques most commonly used to identify the VP4 (P) types of human group A fecal rotaviruses. The techniques included PCR with nested primers and hybridization with PCR-generated probes (to determine the P genotypes). The results obtained by these genetic techniques were evaluated against those obtained by an enzyme immunoassay (EIA) incorporating neutralizing monoclonal antibodies (N-MAbs) reacting with three major human P serotypes (serotypes P1A, P1B, and P2A). The P types of the rotaviruses present in 102 fecal specimens were determined under code by each of the three assays. The specificity of each assay was evaluated against a "gold standard" putative P type (P serotype and genotype) deduced from knowledge of the VP7 (G) type and the origin of the fecal specimen. Overall comparison of the results showed respective sensitivities and specificities of 92 and 92% for reverse transcription-PCR, 80 and 99% for hybridization, and 73 and 91% for EIA with N-MAbs. The hybridization assay retained high sensitivity with specimens stored for > or = 10 years. Hybridization assays with nonradioactive probes are relatively inexpensive and are suited for use in developing countries. In summary, both genetic assays showed high sensitivities and specificities in assigning a P type to human fecal rotavirus strains. Further evaluation of the EIA with N-MAbs is required, together with incorporation of new N-MAbs for the detection of the additional P types detected in developing countries. PMID:9399502

Masendycz, P J; Palombo, E A; Gorrell, R J; Bishop, R F

1997-01-01

168

Highly sensitive microplate chemiluminescence enzyme immunoassay for the determination of staphylococcal enterotoxin B based on a pair of specific monoclonal antibodies and its application to various matrices.  

PubMed

A highly specific and sensitive microplate chemiluminescent enzyme immunoassay (CLEIA) was established and validated for the detection of staphylococcal enterotoxin B (SEB). A pair of monoclonal antibodies (mAbs) that recognizes different epitopes of SEB was selected from 20 SEB-specific mAbs, and the experimental conditions were examined and optimized for the development of the CLEIA. This method exhibited high performance with a dynamic range of 0.01-5 ng/mL, and the measured limit of detection (LOD) was 0.01 ng/mL. Intra- and interassay coefficient variations were all lower than 13% at three concentrations (0.2, 0.4, and 2 ng/mL). For specificity studies, when this method was applied to test staphylococcal enterotoxins A, C1, and D, no cross-reactivity was observed. It has been successfully applied to the analysis of SEB in a variety of environmental, biological and humoral matrices such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, and human urine and serum. The aim of this article is to show that the highly sensitive, specific, and simple microplate CLEIA, based on a pair of highly specific monoclonal antibodies, has potential applications for quantifying SEB in public health and military reconnaissance. PMID:20799707

Liu, Fei; Li, Yongming; Song, Chaojun; Dong, Bangquan; Liu, Zhijia; Zhang, Kui; Li, Haitao; Sun, Yuanjie; Wei, Yuying; Yang, Angang; Yang, Kun; Jin, Boquan

2010-09-15

169

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

PubMed

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

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

2012-11-01

170

Detection of Chlamydia trachomatis in urine samples by nucleic acid tests: comparison with culture and enzyme immunoassay of genital swab specimens.  

PubMed Central

Two commercially available nucleic acid-based tests, ligase chain reaction (LCR; Abbott Laboratories) and PCR (Roche Diagnostics), for the detection of Chlamydia trachomatis in male and female urine samples were compared with culture and enzyme immunoassay (EIA) (Microtrak; Syva) for C. trachomatis detection in genital samples. The samples were collected from 1,005 patients who attended a sexually transmitted disease clinic. In this study population, the prevalence of the infection was 4%. Specimens which were reactive in any of the tests were retested with a different PCR test using primers directed against the major outer membrane protein gene. With a "gold standard" of a positive culture, or any other positive test result if it was confirmed by an independent test, the Roche PCR (95% sensitive, 99.9% specific) was more sensitive than the LCR (75% sensitive, 100% specific) (chi2, P < 0.0001) while both tests were more sensitive than culture (58% sensitive, 100% specific) or EIA (45% sensitive, 100% specific) (chi2, P < 0.001). The Roche PCR and Abbott LCR tests of urine identified 65% and 30% more positive patients, respectively, than did testing by culture of urethral or cervical specimens. Nucleic acid testing of urine specimens for C. trachomatis is a more sensitive and convenient method for the detection of genital infection. PMID:9399559

Schepetiuk, S; Kok, T; Martin, L; Waddell, R; Higgins, G

1997-01-01

171

Evaluation of the diagnostic algorithm consisting of enzyme immunoassay for toxins and polymerase chain reaction, for the diagnosis of Clostridium difficile-associated diarrhoea.  

PubMed

A total of 1631 stool specimens were tested for Clostridium difficile toxins A and B using an enzyme immunoassay (EIA). C. difficile toxin was detected in 191 (11.7%, 191/1631) cases by EIA. Among the remaining 1440 cases, 102 cases in patients with either antibiotic-associated diarrhoea or hospitalized patients with unexplained leukocytosis (>?15,000/mm³) and fever (??38°C) even though they did not meet the criteria for diarrhoea, were further assessed using a polymerase chain reaction (PCR) for the toxin B gene (tcdB). Thirty-four cases were tcdB-positive (33.3%, 34/102). A total of 225 cases (13.8%, 225/1631) had a stool test result positive for C. difficile toxins. Among these, 145 cases were diagnosed with C. difficile-associated diarrhoea (CDAD): 80.7% (117/145) using the EIA and 19.3% (28/145) using the PCR. In our study, the 2-test algorithm including EIA-toxin assay and PCR test made a more accurate diagnosis of CDAD. Furthermore, the application of clinical situations may be effective in the selection of patients who need confirmatory testing. PMID:22813084

Lee, Jae Hoon; Park, Seung Jin; Lee, Yu Min; Lee, Chang Hoon; Cho, Ji Hyun

2012-12-01

172

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

PubMed Central

Serum samples collected from 68 patients (age, mean ± the standard deviation [SD], 56.3 ± 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 ± 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 ± 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection. PMID:15634950

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

2005-01-01

173

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

174

Biotin-avidin amplified enzyme-linked immunosorbent assay (ELISA) for the measurement of canine serum IgA, IgG and IgM  

Microsoft Academic Search

An amplified capture enzyme-linked immunosorbent assay (ELISA) has been developed by the use of the biotin-avidin detection system, for the measurement of canine plasma immunoglobulins (Ig) A, G and M. Test responses of dilutions of both the Ig standards and test plasma samples were consistently linear (r >0·987) for the three Ig classes. The within-assay variation was 3·53 per cent

W. E Bernadina

1996-01-01

175

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

E-print Network

Imnunosorbent Assay. . . . . . . . . . . . . . . . Titration of IgN and IgG in the Enzyme-Linked Immunosorbent Assay. ELISA Results of First Testing of Hybridoma Supernatant Fluids. ELISA Results of Second Test1ng of Hybridoma Supernatant Fluids. ELISA... Results of Third Test1ng of Hybr1doma Supernatant Fluids. ELISA Results of Fourth Testing of Hybri doma Supernatant Fluids. ELISA Results of Fifth Testing of Hybr1doma Supernatant Fluids. Page . . . 16 32 35 36 39 . 43 . 45 . 46 Table IX...

Hunter, Doris Marie

1983-01-01

176

Prevalence of Congenital Toxoplasma gondii Infection among Newborns from the Pozna? Region of Poland: Validation of a New Combined Enzyme Immunoassay for Toxoplasma gondii-Specific Immunoglobulin A and Immunoglobulin M Antibodies  

PubMed Central

We determined the value of a new serological assay detecting Toxoplasma-specific immunoglobulin M (IgM) and IgA antibodies at birth for use in mass neonatal screening. The incidence of congenital infection in newborns was compared with data from an epidemiological investigation on the seroprevalence of Toxoplasma in the studied population. Peripheral blood was collected on Guthrie cards during the first 3 days of life and tested for anti-Toxoplasma IgA and IgM using a noncommercial immunocapture enzyme-linked immunosorbent assay (ELISA). When the screening assay was positive, serum samples from the child and the mother were collected for use in Western blotting comparative immunological profile analysis and traditional serological tests for determination of specific IgG, IgM, and IgA antibodies. From December 1998 to April 2000, 17,653 filter paper samples from live-born neonates were successively screened. Congenital T. gondii infection was finally confirmed in 19 newborns. In traditional assays, 13 of 19 infants were IgM and IgA positive using filter paper eluates at birth, 1 child was positive only for IgM, 1 patient was positive for IgM and borderline for IgA, 1 had an equivocal level of IgA, and 3 cases were confirmed only by the Western blot assay. The prevalence of Toxoplasma-specific IgA and/or IgM in filter paper samples at birth was 1 per 929 live-born neonates (1.08/1,000) or about 1 per 523 children (1.9/1,000) born to nonimmune women with a potential risk of primary T. gondii infection during pregnancy, compared to the actual seropositivity rate of 43.7%. The diagnostic sensitivity of the combined IgA-IgM ELISA using neonatal filter paper specimens was not more than 95%, the positive predictive value of the test was 82.6%, and the diagnostic specificity was calculated to be 99.9%. The combined IgA-IgM ELISA is a valuable method for the diagnosis of congenital toxoplasmosis at birth and fulfills criteria for neonatal screening programs. The method showed a good diagnostic sensitivity in neonates untreated prenatally who were born in an area of high seroprevalence of T. gondii infection. PMID:11326012

Paul, Ma?gorzata; Petersen, Eskild; Szczapa, Jerzy

2001-01-01

177

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

PubMed

Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins (e.g., serum samples) without separation steps is of great significance to biomedical research but remains technically challenging. Here we report a "membrane cloaking" method to overcome nonspecific protein adsorption and fouling problems for label-free surface plasmon resonance detection and heterogeneous immunosensing. A thin, hybrid, self-assembled monolayer on gold was formed with 70 mol % mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles, which fused to form a supported membrane. The analyte spiked in serum was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a nonionic surfactant before the final measurement was carried out. Selection of a suitable surfactant can preserve antibody/antigen binding and selectively remove the membrane, allowing accurate measurement of the captured proteins without interference from nonspecifically adsorbed species. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum ( approximately 75 mg/mL protein) was achieved with the membrane cloaking method, whereas direct measurement without membrane removal resulted in a significantly large error. The cloaking method was also used to develop an enzyme amplified amperometric assay using HRP-conjugated IgG. Detection of concentrations as low as 5 fM proteins was obtained. Finally, a membrane cloaking assay combining SPR and in situ electrochemical measurement was demonstrated on a gold substrate. Similar sensitivity was observed using a continuous flow injection measurement. The method opens new avenues to develop direct assay methods with ultrahigh sensitivity for protein samples using SPR and enzyme-linked amplification mechanisms. PMID:17263314

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

2007-02-01

178

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

179

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

180

New enzyme immunoassay for detection of hepatitis B virus core antigen (HBcAg) and relation between levels of HBcAg and HBV DNA.  

PubMed

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

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

2003-05-01

181

Comparison of Two Commercial Microimmunofluorescence Kits and an Enzyme Immunoassay Kit for Detection of Serum Immunoglobulin G Antibodies to Chlamydia pneumoniae  

PubMed Central

We compared the MRL and the Labsystems Chlamydia pneumoniae microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to C. pneumoniae. The correlation between the results obtained with these two MIF kits was excellent (r = 0.95; P = 0.001). The cross-reactivity of the C. pneumoniae-positive sera with C. trachomatis- and C. psittaci-positive sera was assessed for each MIF kit. For C. pneumoniae-positive sera with titers of ?32, the Labsystems MIF kit exhibited more cross-reactivity to C. psittaci than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to C. pneumoniae (for the MRL MIF kit, r = 0.79 [P = 0.001]; for the Labsystems MIF kit, r = 0.78 [P = 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of C. pneumoniae antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation. PMID:11329463

Messmer, Trudy O.; Martinez, Joseph; Hassouna, Fadwa; Zell, Elizabeth R.; Harris, Wayne; Dowell, Scott; Carlone, George M.

2001-01-01

182

A Highly Sensitive, Multiplex Broad-Spectrum PCR-DNA-Enzyme Immunoassay and Reverse Hybridization Assay for Rapid Detection and Identification of Chlamydia trachomatis Serovars  

PubMed Central

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

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

2007-01-01

183

Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).  

PubMed

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs. PMID:23108105

Ludwig, C; Wachter, B; Silinski-Mehr, S; Ganswindt, A; Bertschinger, H; Hofer, H; Dehnhard, M

2013-01-01

184

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

185

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor II: assay in the plasma and urine of human volunteers by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA).  

PubMed

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. An HPLC-atmospheric-pressure chemical ionisation mass-spectrometric (HPLC-APCI-MS-MS) assay had been already validated (R.F. Venn et al., J. Pharm. Biomed. Anal., in press), but due to its low throughput an alternative method was sought. As the molecule is peptide-like and not metabolised, we believed the immunoassay approach was appropriate. Thus we developed an immunoassay for the compound using time-resolved fluorescence as an end-point (DELFIA) with lower limits of quantification of 0.2 ng ml(-1) for the plasma assay and 5 ng ml(-1) for the assay in urine. This assay is a 96-well plate based competitive immunoassay; the end-point is the determination of a (non-radioactive) europium label by time-resolved fluorimetry. Sampatrilat is labelled with chelated europium via isothiocyanate chemistry. The advantage of this assay is its extremely high throughput, allowing rapid analysis of many thousands of samples. The DELFIA method was successfully cross-validated with the HPLC-APCI-MS-MS method. PMID:9535200

Venn, R F; Barnard, G; Kaye, B; Macrae, P V; Saunders, K C

1998-01-01

186

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

187

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance.  

PubMed

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 microg kg(-1), respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L(-1) phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 microg kg(-1) in liver and 2 microg kg(-1) in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r(2)) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 microg kg(-1)). PMID:18213472

Wang, Lei; Wang, Shuo; Zhang, Jiayi; Liu, Junwei; Zhang, Yan

2008-03-01

188

Immunoassays of soy proteins.  

PubMed

Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine. PMID:12381163

Brandon, David L; Friedman, Mendel

2002-10-23

189

Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay.  

PubMed

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample. PMID:12558032

Ito, Katsutoshi; Nakagawa, Kazuto; Murakami, Seiji; Arakawa, Hidetoshi; Maeda, Masako

2003-01-01

190

Comparison of a novel chemiluminescence enzyme immunoassay (CLEIA) with enzyme-linked immunosorbent assay (ELISA) for the determination of MPO-ANCA in patients with ANCA-associated vasculitis.  

PubMed

Background. Myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) represents the serological hallmark of ANCA-associated vasculitis (AAV). We evaluated the analytical and diagnostic accuracy of chemiluminescence enzyme immunoassay (CLEIA) versus enzyme-linked immunosorbent assay (ELISA) for the detection of MPO-ANCA. Methods. A total of 242 sera obtained from 51 patients with AAV and 103 patients without AAV were tested for MPO-ANCA by ELISA (NephroScholor MPOANC II) and CLEIA (the STACIA MEBLux test). Disease activity in the patients with AAV was determined based on the Birmingham Vasculitis Activity Score. We analyzed the correlations between the MPO-ANCA titers determined by the CLEIA and those determined by the ELISA, and also between the MPO-ANCA titers and the disease activity. Results. The MPO-ANCA titers determined by the CLEIA (x) were strongly correlated with those determined by the ELISA (y). The correlation could be expressed by the following equation in this study: y = 1.8x + 7.7 (r = 0.96; p < 0.0001). At the cutoff value of 3.5 U/ml, the CLEIA yielded positive test results for MPO-ANCA in 73 of the 242 sera (30.2%), while at the cutoff value of 20 U/ml, ELISA yielded positive test results in 57 of the 242 sera (23.6%). The CLEIA yielded false-positive test results in 4 of the 120 sera obtained from the non-AAV patients (3.3%), whereas the ELISA yielded a false-positive result in only 1 of the 120 sera obtained from the non-AAV patients (0.8%). The sensitivity and specificity of the CLEIA for the diagnosis of AAV were 100% and 96.7%, respectively, while those of the ELISA were 94.3% and 99.2%, respectively. The sensitivity and specificity of the CLEIA for the prediction of active disease were 100% and 64.4%, respectively, while those of the ELISA were 94.3% and 73.6%, respectively. Conclusion. The false positivity rate of the CLEIA for MPO-ANCA tended to be high as compared with that of the ELISA. Also, according to the correlation coefficient between the results of the CLEIA and the ELISA calculated in this study, it is necessary to pay attention to the differences in the sensitivity and specificity between CLEIA and ELISA. PMID:25388618

Hirose, Orie; Itabashi, Mitsuyo; Takei, Takashi; Nitta, Kosaku

2014-11-12

191

Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada.  

PubMed

Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producing Escherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples. PMID:25588656

Chui, Linda; Patterson-Fortin, Laura; Kuo, Julie; Li, Vincent; Boras, Valerie

2015-03-01

192

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

E-print Network

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

Ball, Madalyn Eunice

1993-01-01

193

Microbead-based electrochemical immunoassay with interdigitated array electrodes  

Microsoft Academic Search

The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-?m gaps and 2.4-?m widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse

Jennifer H Thomas; Sang Kyung Kim; Peter J Hesketh; H. Brian Halsall; William R Heineman

2004-01-01

194

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 ...

195

A point-of-care test for measles diagnosis: detection of measles-specific IgM antibodies and viral nucleic acid  

PubMed Central

Abstract Objective To evaluate the performance of a newly developed point-of-care test (POCT) for the detection of measles-specific IgM antibodies in serum and oral fluid specimens and to assess if measles virus nucleic acid could be recovered from used POCT strips. Methods The POCT was used to test 170 serum specimens collected through measles surveillance or vaccination programmes in Ethiopia, Malaysia and the Russian Federation: 69 were positive for measles immunoglobulin M (IgM) antibodies, 74 were positive for rubella IgM antibodies and 7 were positive for both. Also tested were 282 oral fluid specimens from the measles, mumps and rubella (MMR) surveillance programme of the United Kingdom of Great Britain and Northern Ireland. The Microimmune measles IgM capture enzyme immunoassay was the gold standard for comparison. A panel of 24 oral fluids was used to investigate if measles virus haemagglutinin (H) and nucleocapsid (N) genes could be amplified by polymerase chain reaction directly from used POCT strips. Findings With serum POCT showed a sensitivity and specificity of 90.8% (69/76) and 93.6% (88/94), respectively; with oral fluids, sensitivity and specificity were 90.0% (63/70) and 96.2% (200/208), respectively. Both H and N genes were reliably detected in POCT strips and the N genes could be sequenced for genotyping. Measles virus genes could be recovered from POCT strips after storage for 5 weeks at 20–25 °C. Conclusion The POCT has the sensitivity and specificity required of a field-based test for measles diagnosis. However, its role in global measles control programmes requires further evaluation. PMID:21897488

Slibinskas, Rimantas; Chua, Kaw Bing; Nigatu, Wondatir; Brown, Kevin E; Sasnauskas, Kestutis; Samuel, Dhanraj; Brown, David

2011-01-01

196

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

197

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

198

COUPLING OF IMMUNOASSAYS WITH ENZYMATIC RECYCLING ELECTRODES  

Microsoft Academic Search

Enzymatic substrate regeneration is a tool to enhance the sensitivity of enzyme electrodes both for substrate analysis and immunoassays. The combination of immunoreactions and electrode based substrate recycling connects specific recognition of an analyte with highly sensitive detection. Most important for this field of application is the sensitivity, which permits to detect a label at very low concentration. Enzymatic substrate

Frieder W. Scheller; Christian G. Bauer; Alexander Makower; Ulla Wollenberger; Axel Warsinke; Frank F. Bier

2001-01-01

199

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

PubMed Central

Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR?/? mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

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

2012-01-01

200

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

201

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

PubMed Central

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

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

2013-01-01

202

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

203

Comparison of Surface Plasmon Resonance, Resonant Waveguide Grating Biosensing and Enzyme Linked Immunosorbent Assay (ELISA) in the Evaluation of a Dengue Virus Immunoassay  

PubMed Central

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.

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

204

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

205

Comparison of dissociation-enhanced lanthanide fluorescent immunoassays to enzyme-linked immunosorbent assays for detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus.  

PubMed

The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log(10) concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

Smith, D R; Rossi, C A; Kijek, T M; Henchal, E A; Ludwig, G V

2001-11-01

206

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

PubMed Central

The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

2001-01-01

207

Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe3O4/polyaniline platform and multi-enzyme-functionalized carbon sphere label.  

PubMed

An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe(3)O(4)/polyaniline/Nafion (Fe(3)O(4)/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab(2)) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe(3)O(4)/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe(3)O(4)/polyaniline (Fe(3)O(4)/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8 pM and 2 nM with the detection limit of 4 pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants. PMID:22444540

Lin, Mouhong; Liu, Yingju; Sun, Zihong; Zhang, Shenglai; Yang, Zhuohong; Ni, Chunlin

2012-04-13

208

A rapid, near infrared, whole blood immunoassay using metal nanoshells  

Microsoft Academic Search

The development of a rapid, whole blood immunoassay with the capacity to detect various analytes would greatly benefit point-of-care or public health applications, where there is a strong demand for the rapid, high throughput screening of blood-borne species. Immunoassays, with their high affinity\\/specificity, show the most promise for implementing such a system. Nevertheless, conventional methods, such as the enzyme linked

L. R. Hirsch; N. J. Halas; J. L. West

2002-01-01

209

Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA.  

PubMed

The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The specificity, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the 'false positive' results characterised by very weak bands could be discounted by re-examination of the results at 60 min, yielding a specificity of 100%. With this minor modification of the procedure applied to examination of urine samples from 117 patients with legionellosis confirmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases confirmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was significantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid specific test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously. PMID:11393288

Helbig, J H; Uldum, S A; Lück, P C; Harrison, T G

2001-06-01

210

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

211

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

212

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery.  

PubMed

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers. PMID:11829670

Thirumala-Devi, K; Mayo, M A; Hall, A J; Craufurd, P Q; Wheeler, T R; Waliyar, F; Subrahmanyam, A; Reddy, D V R

2002-02-13

213

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

214

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

215

Early Neonatal Diagnosis of Congenital Toxoplasmosis: Value of Comparative Enzyme-Linked Immunofiltration Assay Immunological Profiles and Anti-Toxoplasma gondiiImmunoglobulin M (IgM) or IgA Immunocapture and Implications for Postnatal Therapeutic Strategies  

Microsoft Academic Search

Diagnostic strategies for congenital toxoplasmosis have changed profoundly in recent years. Immunological diagnosticmethods,longconsidereddisappointing,cannowbeusedataveryearlystage.Overa3-yearperiod, 1,050 infants at risk of congenital toxoplasmosis (born to 1,048 mothers infected during pregnancy) were monitored for a minimum of 12 months and a maximum of 7 years. More than 6,000 serum specimens were analyzed by comparative mother-infant immunological profiles (CIPs) based on an enzyme-linked immuno- filtrationassay(ELIFA)andanimmunocapturemethodforthedetectionofspecificimmunoglobulinM(IgM)

J. M. PINON; C. CHEMLA; I. VILLENA; F. FOUDRINIER; D. AUBERT; D. PUYGAUTHIER-TOUBAS; B. LEROUX; D. DUPOUY; C. QUEREUX; M. TALMUD; T. TRENQUE; G. POTRON; M. PLUOT; G. REMY; Robert Debre ´

216

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

Microsoft Academic Search

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

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

1986-01-01

217

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

PubMed Central

West Nile (WN) virus was introduced into the United States in 1999, when the first human cases of WN fever and encephalitis appeared in New York City. From there, the virus has spread throughout North America, in some areas cocirculating with the related flavivirus St. Louis encephalitis (SLE) virus. Public health laboratories currently use an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) as a primary test for human serodiagnosis, followed by a confirmatory plaque-reduction neutralization test (PRNT). The MAC-ELISAs take 2 days to perform; therefore there is a need for a more rapid test. This report describes a duplex microsphere-based immunoassay (MIA) that shortens the test processing time to about 4.5 h. The assay employs two sets of microspheres coupled to a single flavivirus group-reactive antibody, which are used to capture the WN and SLE viral antigens independently. Immunoglobulin G-depleted serum is concurrently assayed for IgM antibodies to each of the viral antigens. The results are standardized and classified by using quadratic discriminant analysis so that a single result, anti-WN IgM-positive, anti-SLE IgM-positive, negative, or nonspecific, can be determined. The duplex MIA results compared favorably to those of the plaque-reduction neutralization test and MAC-ELISA. The assay proved to be reproducible, produced accurate classifications as to the infecting virus, and was specific. PMID:15879016

Johnson, Alison J.; Noga, Amanda J.; Kosoy, Olga; Lanciotti, Robert S.; Johnson, Alicia A.; Biggerstaff, Brad J.

2005-01-01

218

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

219

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

220

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

221

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.

222

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

PubMed Central

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis. PMID:17609393

Johnson, Alison J.; Cheshier, Ronald C.; Cosentino, Giorgio; Masri, Heather P.; Mock, Valerie; Oesterle, Rebecca; Lanciotti, Robert S.; Martin, Denise A.; Panella, Amanda J.; Kosoy, Olga; Biggerstaff, Brad J.

2007-01-01

223

Metal-enhanced immunoassays.  

PubMed

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

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

2012-01-01

224

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

225

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

226

Lanthanide-based time-resolved luminescence immunoassays.  

PubMed

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized. PMID:21556751

Hagan, A K; Zuchner, T

2011-07-01

227

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

PubMed

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

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

2001-08-01

228

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

229

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers.  

PubMed

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3?. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method. PMID:22355804

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

2012-04-01

230

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

231

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

232

Determination of Designer Drug Cross-Reactivity on Five Commercial Immunoassay Screening Kits.  

PubMed

The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), ?-keto amphetamines, substituted amphetamines, piperazines, ?-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits. PMID:25492523

Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z

2014-12-01

233

A highly efficient colorimetric immunoassay using a nanocomposite entrapping magnetic and platinum nanoparticles in ordered mesoporous carbon.  

PubMed

Nanocomposite to achieve ultrafast immunoassay: a new synergistically integrated nanocomposite consisting of magnetic and platinum nanoparticles, simultaneously entrapped in mesoporous carbon, is developed as a promising enzyme mimetic candidate to achieve ultrafast colorimetric immunoassays. Using new assay system, clinically important target molecules, such as human epidermal growth factor receptor 2 (HER2) and diarrhea-causing rotavirus, can be detected in only 3 min at room temperature with high specificity and sensitivity. PMID:23832855

Kim, Moon Il; Ye, Youngjin; Woo, Min-Ah; Lee, Jinwoo; Park, Hyun Gyu

2014-01-01

234

A Functional Protein S and Microlatex Immunoassay for Protein S and C4b-Binding Protein on the Automated Coagulation Laboratory (ACL) 300 Plus  

Microsoft Academic Search

Protein S can be determined by functional or immunological assays. Electroimmunodiffusion (EID) or enzyme immunoassays (enzyme-linked immunosorbent assay; ELISA) are the commonly employed techniques for measuring protein S and C4b-binding protein (C4b- BP) immunologically. Procedures for these assays are time-consuming and labor-intensive. The introduction of microlatex immunoassays (LIATEST system; Diagnos tica Stago, Asnieres-Sur-Seine, France) has provided an alternative for rapid

Shinichiro Hirokawa; Eberhard F. Mammen

1996-01-01

235

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

with sera from S19 inoculated cattle but not with serum from a Strain 2308 infected cow. An Ig'VI antibody class-specific enzyme-linked immunosorbent assay (ELISA) was performed on sera from Strain 19 or 2308 exposed cattle, using 'T24' as the antigen... phase. Statistical analysis of ELISA results placed some Strain 19 and 2308 exposed cattle into separate and statistically (at the 0. 01~& level) different populations. ACKNOWLEDGEMENTS The author would like to thank Drs. K. H. Nielsen, F. C. Heck...

Smithwick, Charles Ripley

1983-01-01

236

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...

237

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...

238

A homogeneous chemiluminescent immunoassay method.  

PubMed

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

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

2013-03-20

239

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

240

IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens.  

PubMed

IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans. PMID:25320238

Maglione, Paul J; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R; Bagiella, Emilia; Bussel, James B; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine; Cunningham-Rundles, Charlotte

2014-12-01

241

Invalidation of a commercially available human 5?-dihydrotestosterone immunoassay.  

PubMed

Enzyme immunoassays (EIA) are commonly utilized for the evaluation of androgens in biological fluids; however, careful consideration must be given to cross-reactivity with other endogenous sex-steroid hormones. Our purpose was to determine the validity of a commonly-utilized commercially-available dihydrotestosterone (DHT) EIA. Serum samples obtained from older hypogonadal men who participated in a 12-month randomized controlled trial evaluating the effects of testosterone-enanthate (125 mg/week) or vehicle in combination with finasteride (5mg/day) or placebo were assayed for DHT via EIA and using a validated gold-standard LC-MS/MS approach. Additionally, commercially-available (DHT-free) buffer containing graded testosterone doses was evaluated by DHT immunoassay. DHT concentrations measured via EIA were 79% to >1000% higher than values obtained by LC-MS/MS (p<0.05), with the largest differences (415-1128%) occuring in groups receiving finasteride. Both LC-MS/MS and EIA indicated that testosterone-enanthate increased serum DHT to a similar magnitude. In contrast, finasteride-induced reductions in DHT were detected by LC-MS/MS, but not EIA (p<0.05). No significant associations were present for DHT concentrations between measurement techniques. Cross-reactivity of testosterone with the immunoassay ranged from 18% to 99% and DHT concentrations measured by EIA were highly associated with the spiked testosterone concentrations in DHT-free buffer (r=0.885, p<0.001). In conclusion, we provide evidence invalidating a commonly-utilized commercially-available DHT immunoassay because significant cross-reactivity exists between testosterone and the EIA and because the changes in DHT observed via EIA were not associated with a validated gold-standard measurement technique. The cross-reactivity of testosterone is particularly concerning because testsoterone is present in 100-fold greater concentrations than is DHT within the circulation. PMID:24012740

Yarrow, Joshua F; Beck, Darren T; Conover, Christine F; Beggs, Luke A; Goldberger, Bruce A; Borst, Stephen E

2013-12-11

242

IGM heating in fossil galaxy groups  

NASA Astrophysics Data System (ADS)

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

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

2014-10-01

243

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...

244

Isotope-labeled immunoassays without radiation waste  

E-print Network

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

Hammock, Bruce D.

245

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

PubMed

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

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

1995-06-01

246

Mass loss from galaxies: feeding the IGM, recycling in the IGM  

E-print Network

As a result of internal processes or environmental effects like ram-pressure stripping or collisions, galaxies lose a significant part of their stellar and gaseous content. Whereas the impact of such stripping on galaxy evolution has been well studied, much less attention has been given to the fate of the expelled material in the intergalactic or intracluster medium (IGM/ICM). Observational evidence exists showing that a fraction of the injected matter is actually recycled to form a new generation of galaxies, such as the Tidal Dwarf Galaxies discovered near numerous interacting systems. Using a set of multiwavelength data, we are now able to roughly analyze the processes pertaining to their formation: from an instability in the HI clouds, through the formation of molecular gas, and to the onset of star formation.

Pierre-Alain Duc; Jonathan Braine; Ute Lisenfeld; Philippe Amram; Elias Brinks

2001-11-30

247

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

248

Rapid detection of cardioactive bufalin toxicity using fluorescence polarization immunoassay for digitoxin.  

PubMed

Intoxication caused by digitalis-like substances after ingestion of cooked toad soup has been reported. Bufalin, a cardioactive compound, is found in toad. Bufalin is also found in many Chinese medicines. Earlier reports demonstrated cross reactivity of bufalin with fluorescence polarization immunoassay for digoxin. In this report, the authors demonstrated a significantly higher cross reactivity of bufalin with the fluorescence polarization assay for digitoxin. They supplemented aliquots of normal plasma that had various concentrations of bufalin (1 to 50 micrograms/ml) from a local blood bank and measured apparent digitoxin concentrations using fluorescence polarization immunoassay and chemiluminescent assays (ACS digitoxin) for digitoxin. They measured apparent digoxin and digitoxin concentrations using fluorescence polarization, microparticle enzyme immunoassay, and chemiluminescent assays for digitoxin. They observed apparent digitoxin or digoxin concentrations in sera supplemented with bufalin only with the fluorescence polarization assays. For example, the apparent digitoxin concentration observed in a serum supplemented with 25 ng/ml of bufalin was 24.3 ng/ml of digitoxin equivalent. The apparent digoxin concentration observed in the same specimen was 1.33 ng/ml digoxin equivalent. Bufalin caused positive interference in serum digoxin or digitoxin measurements in specimens containing digoxin or digitoxin when concentrations were measured by fluorescence polarization assays. In contrast, bufalin lowered the measured digoxin concentrations in serum pools containing digoxin when digoxin concentrations were measured by the microparticle enzyme immunoassay. The authors conclude that bufalin toxicity can be rapidly detected by the fluorescence polarization assay for digitoxin. PMID:9485564

Dasgupta, A; Datta, P

1998-02-01

249

Successful treatment of IgM paraproteinaemic neuropathy with fludarabine  

PubMed Central

OBJECTIVES—To evaluate the response of four patients with IgM paraproteinaemic neuropathy to a novel therapy—pulsed intravenous fludarabine.?BACKGROUND—The peripheral neuropathy associated with IgM paraproteinaemia usually runs a chronic, slowly progressive course which may eventually cause severe disability. Treatment with conventional immunosuppressive regimens has been unsatisfactory. Fludarabine is a novel purine analogue which has recently been shown to be effective in low grade lymphoid malignancies.?METHODS—Four patients were treated with IgM paraproteinaemic neuropathy with intravenous pulses of fludarabine. Two of the four patients had antibodies to MAG and characteristic widely spaced myelin on nerve biopsy and a third had characteristic widely spaced myelin only. The fourth had an endoneurial lymphocytic infiltrate on nerve biopsy and a diagnosis of Waldenström's macroglobulinaemia.?RESULTS—In all cases subjective and objective clinical improvement occurred associated with a significant fall in the IgM paraprotein concentration in three cases. Neurophysiological parameters improved in the three patients examined. The treatment was well tolerated. All patients developed mild, reversible lymphopenia and 50% mild generalised myelosuppression, but there were no febrile episodes.?CONCLUSION—Fludarabine should be considered as a possible treatment for patients with IgM MGUS paraproteinaemic neuropathy.?? PMID:10209166

Wilson, H.; Lunn, M.; Schey, S.; Hughes, R

1999-01-01

250

Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus.  

PubMed

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

2011-06-01

251

Plasmonic Technology: Novel Approach to Ultrasensitive Immunoassays  

PubMed Central

At the Center for Fluorescence Spectroscopy, we have taken advantage of the favorable properties of surface plasmon-coupled emission (SPCE) to improve fluorescence-based immunoassays. SPCE occurs when excited fluorophores near conducting metallic structures efficiently couple to surface plasmons. These surface plasmons, appearing as free electron oscillations in the metallic layer, produce electromagnetic radiation that preserves the spectral properties of fluorophores but is highly polarized and directional. SPCE immunoassays provide several advantages over other fluorescence-based methods. This review explains new approaches to fluorescence immunoassays, including our own use of SPCE for simultaneous detection of more than one fluorescent marker and performance of immunoassays in the presence of an optically dense medium, such as whole blood. PMID:16055432

Lakowicz, Joseph R.; Malicka, Joanna; Matveeva, Evgenia; Gryczynski, Ignacy; Gryczynski, Zygmunt

2009-01-01

252

Heterogeneous and homogeneous immunoassays for drug analysis.  

PubMed

Immunoassays are very useful techniques to perform screening and semi-quantitative analysis of hundreds of different xenobiotics. Small sample volumes are required and pretreatment is usually unnecessary (e.g., homogeneous immunoassays). Fully automated and high-throughput systems are available, which help physicians to take timely decisions. However, immunoassays do suffer from interference from both endogenous and exogenous factors that limit their application in quantitative analysis. These assays use different labels (e.g., colorimetric, fluorescent, chemiluminescent or electrochemiluminescent) and different methods for generating and measuring signals, but the basic principles are usually similar. This review outlines the practical aspects of immunoassays in bioanalysis and describes their application in clinical chemistry for xenobiotic analysis, namely medicines and drugs of abuse. PMID:25486233

Dinis-Oliveira, Ricardo Jorge

2014-11-01

253

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor  

Microsoft Academic Search

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)25 (DBT) with

E Pale?ek; R Kizek; L Havran; S Billova; M Fojta

2002-01-01

254

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

255

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

256

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

PubMed

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

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

2011-07-01

257

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 ...

258

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...

259

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

E-print Network

to about 60 min. Introduction Immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), have become one of the most powerful biochemical analysis techniques.1,2 Traditionally, these assays employing various detec- tions methods such as fluorometric and colorimetric measure- ment, thermal lensing

Zare, Richard N.

260

Recombinant hepatitis A virus antigen: improved production and utility in diagnostic immunoassays.  

PubMed

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

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

1998-07-01

261

Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs  

Microsoft Academic Search

An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of

M. Subathra; T. M. A. Senthilkumar; P. Ramadass; G. Dhinakar Raj

2011-01-01

262

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

263

Sensitive chemiluminescent immunoassay of triclopyr by digital image analysis.  

PubMed

An image based detection of chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the quantification of triclopyr has been developed. The immunoassay was an indirect competitive immunoassay with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence was produced by the luminol/H(2)O(2)/HRP reaction, detected by a monochrome video CCD camera and digitized with an Imagraph IC-PCI frame grabber using a custom program developed in C(++) (Microsoft Visual C(++) 6.0). Two main improvements are reported in the data processing software: the implementation of a circular mesh covering the perimeter of each well, eliminating diffuse light from the neighboring wells, and the use of volume (the integration of light intensity of all pixels that define a well) as an analytical signal instead of CL intensity or area (as usual in commercial plate readers) to improve precision for normalization of the total light output. The standard curve was produced for 0.01-10 ng/L triclopyr. The limit of detection was 0.8 ng/L and the variation coefficient was 3.07% (n=10, P=0.05). PMID:22841045

Díaz, Aurora N; Sánchez, Francisco G; Baro, Enrique N; Díaz, Ana F G; Aguilar, Alfonso; Algarra, Manuel

2012-08-15

264

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

PubMed

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

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

2014-04-01

265

Solitary IgM phase II response has a limited predictive value in the diagnosis of acute Q fever.  

PubMed

We investigated the positive predictive value (PPV) of a solitary positive immunoglobulin M (IgM) phase II response for the serodiagnosis of acute Q fever detected with either an indirect immunofluorescence assay (IFA) or an enzyme-linked immunosorbent assay (ELISA). Initial and follow-up sera from patients suspected of acute Q fever were included if initially only IgM phase II tested positive with IFA in 2008 (n=92), or ELISA in 2009 (n=85). A seroconversion for Q fever was defined as an initial sample being IgG phase II negative but positive in the follow-up sample. The PPV of an initial isolated IgM phase II result detected by IFA or ELISA was 65% and 51%, respectively, and therefore appeared not to adequately predict acute Q fever. For this reason it cannot be used as a diagnostic criterion nor should it be included in public health notification without confirmation with other markers or a follow-up serum sample. PMID:22340504

Raven, C F H; Hautvast, J L A; Herremans, T; Leenders, A C A P; Schneeberger, P M

2012-11-01

266

Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies  

Microsoft Academic Search

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26kDa, respectively. Anti-fish IgM antibody against all the three species were

Manas Ranjan Bag; M. Makesh; K. V. Rajendran; S. C. Mukherjee

2009-01-01

267

Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody.  

PubMed

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

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

2014-10-01

268

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

269

Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand.  

PubMed

We compared the performance of 2 commercially available dipstick assays, 2 enzyme-linked immunosorbent assays (ELISAs), and an indirect immunofluorescent antibody (IFA) assay for the diagnosis of scrub typhus, using the indirect immunoperoxidase (IIP) test as the reference standard. The dipstick assays were the Integrated Diagnostics (Baltimore, MD) Dip-S-Ticks Scrub Recombinant (r56) dipstick test (INDX assay) and the PanBio (Brisbane, Australia) Scrub Typhus IgM and IgG Rapid Immunochromatographic test (PanBio assay). One of the ELISAs used pooled cell lysates of Karp, Kato, and Gilliam strain Orientia tsutsugamushi as antigen (pooled-antigen ELISA), and the other used a recombinant r56 protein as the antigen (recombinant ELISA). With a panel of 123 positive and 227 negative sera, sensitivity and specificity of the assays were as follows: INDX assay, IgG, 60% and 95%, IgM, 60% and 97%; PanBio assay, IgG, 94% and 96%, IgM, 83% and 93%; IFA (1:400 cutoff), IgG, 91% and 96%, IgM, 85% and 98%; pooled-antigen ELISA, IgG (1:1600 cutoff), 97% and 89%, IgM (1:400 cutoff), 94% and 91%; recombinant ELISA, IgG (1:1600 cutoff), 97% and 92%, IgM (1:400 cutoff), 93% and 94%. Because of its excellent performance and use of a standardized, commercially available antigen, the recombinant ELISA is suitable for use in a diagnostic laboratory, where it may be able to replace the IFA and IIP assays. In contrast, the PanBio dipstick assay was easy to perform and did not require sophisticated equipment, making it suitable for use in rural areas where more sophisticated diagnostic tests such as the ELISA and IFA may not be available. PMID:12479551

Coleman, Russell E; Sangkasuwan, Vichai; Suwanabun, Nantavadee; Eamsila, Chirapa; Mungviriya, Siriporn; Devine, Peter; Richards, Allen L; Rowland, Denise; Ching, Wei-Mei; Sattabongkot, Jetsumon; Lerdthusnee, Kriangkrai

2002-11-01

270

Thermal diffusion in the IGM of clusters of galaxies  

E-print Network

We revisit the phenomenon of elements diffusion in the intergalactic medium (IGM) in clusters of galaxies. The diffusion is driven by gravity, concentration and temperature gradients. The latter cause thermal diffusion, which has been so far ignored in IGM studies. We consider the full problem based on the Burgers' equations and demonstrate that the temperature gradients present in clusters of galaxies may successfully compete with gravity, evacuating metals from cooler regions. Under the combined action of gravity and temperature gradients, complicated metallicity profiles with several peaks and depressions may be formed. For a typical cool core cluster, the thermal diffusion may significantly reduce and even reverse the gravitational sedimentation of metals, resulting in the depression in their abundance in the core. This may have implications for diagnostics of the low temperature plasma in the centers of clusters of galaxies.

Shtykovskiy, P

2009-01-01

271

Natural IgM and IgG Autoantibodies to Epidermal Keratins in Normal Human Sera. I: ELISA-Titration, Immunofluorescence Study  

Microsoft Academic Search

This paper presents a study of autoantibodies (autoAB) to keratins and to epidermis by a double approach associating a specific immunoenzymatic technique and immunofluorescence. The existence of natural autoAB to keratins in all normal human sera was asserted and the heterogeneity of natural autoAB to the epidermis explored. By a sensitive enzyme-linked immunosorbent assay we detected natural IgM and IgG

Guy Serre; Christian Vincent; Roland Viraben; Jean-Pierre Soleilhavoup

1987-01-01

272

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer.  

PubMed

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method. PMID:18220412

Tang, Dianping; Yuan, Ruo; Chai, Yaqin

2008-03-01

273

IgM, Fc?-receptors and malarial immune evasion  

PubMed Central

"This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org." Immunoglobulin M (IgM) is an ancestral antibody class found in all jawed vertebrates from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied, and most enigmatic of the vertebrate immunoglobulins has recently been shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. Here we discuss how this association might come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just discovered Fc?-receptor (FcµR) localized to B and T cell surfaces. As this parasite may exploit an interaction with IgM to not only limit immune detection but also manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies. PMID:20410497

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

2010-01-01

274

Immunoassay, biosensors and other nonchromatographic methods  

E-print Network

based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selec- tively bound malathion.4 A few years later, radioimmunoassays were developed for the detection of products containing genetically modified organisms (GMOs). The advantages of immunoassay

Hammock, Bruce D.

275

Laser Light Scattering Immunoassay for Malaria  

Microsoft Academic Search

Laser light scattering immunoassay (LIA) was proposed as a prospective diagnostic method for the detection of antibody (or antigen) by monitoring the agglutination of antigen (or antibody) coated carrier particles using dynamic light scattering (DLS) as probe, LIA is a very sensitive assay as it can detect microscopic immune complexes even when antibody (or antigen) level is low. A sizeable

Priyaranjan Bhakat; Arati Roy; Kunal B. Roy; Anita Saxena; Himadri B. Bohidar

1999-01-01

276

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...

277

Monitoring human exposure to pesticides using immunoassay  

E-print Network

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

Hammock, Bruce D.

278

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

279

Hapten modification approach for switching immunoassay specificity from selective to generic.  

PubMed

The cross-reactivity profile of polyclonal antibodies against a low molecular weight analyte is strongly influenced by design of the coating or enzyme-linked hapten. The hapten modification effect on immunoassay specificity was studied. Heterology in hapten type and linking method were applied. The influence of these factors on analyses of two groups of antibiotics, 16-membered macrolides and glycopeptides was studied. This approach was used to convert the selective ELISAs to tylosin and eremomycin for group determination of tylosin\\tilmicosin, tylosin\\spiramycin and eremomycin\\vancomycin. It was shown that the analytical spectrum of the developed polyclonal antibody-based immunoassays could be expanded and depended mainly on the type of coating hapten but not on the linking method. Modification of the hapten type in coating conjugates applied in present study served as a mechanism for switching specificity of the ELISA between selective and group. PMID:23234756

Burkin, Maksim A; Galvidis, Inna A

2013-02-28

280

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

281

Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies.  

PubMed

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM. PMID:19063976

Bag, Manas Ranjan; Makesh, M; Rajendran, K V; Mukherjee, S C

2009-02-01

282

Production of IgM specific recombinant dengue multiepitope protein for early diagnosis of dengue infection.  

PubMed

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost. PMID:17256968

Tripathi, Nagesh K; Shrivastva, Ambuj; Pattnaik, Priyabrata; Parida, Manmohan; Dash, Paban K; Gupta, Nimesh; Jana, Asha M; Rao, P V Lakshmana

2007-01-01

283

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

PubMed

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

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

2013-01-01

284

Industrial Fungal Enzymes: An Occupational Allergen Perspective  

PubMed Central

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

Green, Brett J.; Beezhold, Donald H.

2011-01-01

285

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. ...

286

TSE Diagnostics: Recent Advances in Immunoassaying Prions  

PubMed Central

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone, ?-sheet-rich isoform, named prion. Definite diagnosis of TSEs is determined post mortem. For this purpose, immunoassays for analyzing brain tissue have been developed. However, the ultimate goal of TSE diagnostics is an ante mortem test, which would be sensitive enough to detect prions in body fluids, that is, in blood, cerebrospinal fluid, or urine. Such a test would be of paramount importance also for screening of asymptomatic carriers of the disease with the aim of increasing food, drugs, and blood-derived products safety. In the present paper, we have reviewed recent advances in the development of immunoassays for the detection of prions. PMID:23970925

Lukan, Anja; Vranac, Tanja; ?urin Šerbec, Vladka

2013-01-01

287

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

288

Southern elephant seals: IgM concentration in milk of cows and serum of their pups  

Microsoft Academic Search

Serum and milk Immunoglobulin M (IgM) concentrations in 11 mother-pup pairs were measured in southern elephant seals (Mirounga leonina) throughout lactation during 2 breeding seasons at King George Island. Samples were obtained sequentially throughout the\\u000a suckling period (approximately 23?days). The IgM concentration was measured by single radial immunodiffusion on agarose plates.\\u000a Milk IgM concentrations showed significant differences throughout lactation, with

Maria E. I. Marquez; A. R. Carlini; A. V. Baroni; N. H. Slobodianik; P. A. Ronayne de Ferrer; M. F. Godoy

2000-01-01

289

Radio Galaxies and the Magnetization of the IGM  

E-print Network

Observed radio galaxies had a much higher comoving density during the `quasar era', at z ~ 2-3, but these sources are only detectable for small fractions of their active lifetimes at such high z due to expansion losses and increased inverse Compton losses against the cosmic microwave background. Using recent models for the evolution of the size and luminosity of powerful double radio sources, as well as LCDM simulations of the cosmic web of baryonic material, we argue that during the quasar era a high volume fraction of this web was occupied by the lobes of double radio sources. They could have seeded the IGM with an average magnetic field approaching 10^{-8} G. Further, these advancing overpressured lobes could compress the denser interstellar gas clouds of the galaxies engulfed by them and thus trigger starbursts. This can probably account for much of the intense star-formation activity witnessed beyond z ~ 1.5. Also, the sweeping up of the ISM of the gas-rich galaxies by the rapidly advancing radio lobes may well be responsible for the widespread metal pollution of the IGM and proto-galaxies at high redshifts.

Gopal-Krishna; Paul J. Wiita

2003-01-15

290

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

291

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

292

Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement.  

PubMed

A successively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent enlargement by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibody. After all the processes comprising the formation of immunocomplex, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1-10 ng ml(-1) with a detection limit of 0.03 ng ml(-1). Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays. PMID:17720472

Chen, Zhao-Peng; Peng, Zhao-Feng; Luo, Yan; Qu, Bo; Jiang, Jian-Hui; Zhang, Xiao-Bing; Shen, Guo-Li; Yu, Ru-Qin

2007-11-30

293

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

294

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

295

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

296

Magnetic electrochemical immunoassays with quantum dot labels for detection of phosphorylated acetylcholinesterase in plasma.  

PubMed

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. PMID:18855408

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

2008-11-15

297

Two Distinct Subtypes of Hepatitis B Virus-Related Acute Liver Failure Are Separable By Quantitative Serum IgM anti-HBc and HBV DNA Levels  

PubMed Central

Background Hepatitis B virus-related acute liver failure (HBV-ALF) may occur following acute HBV infection (AHBV-ALF) or during an exacerbation of chronic HBV infection (CHBV-ALF). Clinical differentiation of the two is often difficult if a prior history of hepatitis B is not available. Quantitative measurements of anti-hepatitis B core immunoglobulin M (IgM anti-HBc) titers and of HBV viral loads (VLs) might allow separation of acute from chronic HBV-ALF. Methods Of 1602 patients with ALF, 60 met clinical criteria for AHBV-ALF and 27 for CHBV-ALF. Sera were available on 47 and 23 patients, respectively. A quantitative immunoassay was used to determine IgM anti-HBc levels, and real-time polymerase chain reaction (rtPCR) to determine HBV VLs. Results AHBV-ALFs had much higher IgM anti-HBc titers than CHBV-ALFs, (signal to noise (S/N) ratio median 88.5, range 0–1,120, vs. 1.3, 0–750, p<0.001); a cut point for S/N ratio of 5.0 correctly identified 44/46 (96%) AHBV-ALFs and 16/23 (70%) CHBV-ALFs; the area under the receiver operator characteristic curve was 0.86, p<0.001. AHBV-ALF median admission VL was 3.9 (0–8.1) log10 IU/mL, vs. 5.2 (2.0–8.7) log10 IU/mL for CHBV-ALF, p<0.025. Twenty percent (12/60) of the AHBV-ALF group had no hepatitis B surface antigen (HBsAg) detectable on admission to study, while no CHBV-ALF patients experienced HBsAg clearance. Rates of transplant-free survival were 33% (20/60) for AHBV-ALF vs. 11% (3/27) for CHBV-ALF, p=0.030. Conclusions AHBV-ALF and CHBV-ALF differ markedly in IgM anti-HBc titers, in HBV VLs and in prognosis, suggesting that the two forms are indeed different entities that might each have a unique pathogenesis. PMID:21987355

Dao, Doan Y; Hynan, Linda S.; Yuan, He-Jun; Sanders, Corron; Balko, Jody; Attar, Nahid; Lok, Anna S.F.; Word, R. Ann; Lee, William M.

2011-01-01

298

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

299

Development of a New Cytomegalovirus (CMV) Immunoglobulin M (IgM) Immunoblot for Detection of CMV-Specific IgM  

Microsoft Academic Search

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used

T. LAZZAROTTO; A. RIPALTI; G. BERGAMINI; M. C. BATTISTA; P. SPEZZACATENA; F. CAMPANINI; P. PRADELLI; S. VARANI; L. GABRIELLI; G. T. MAINE; M. P. LANDINI

1998-01-01

300

Evidence of fibrinogen as a target of citrullination in IgM rheumatoid factor-positive polyarticular juvenile idiopathic arthritis  

PubMed Central

Background Several studies have noted the significance of measuring anti-cyclic citrullinated peptide (CCP) antibodies in juvenile idiopathic arthritis (JIA) as an important indicator for destructive disease, as is the case in rheumatoid arthritis (RA). While the role of anti-CCP antibodies in RA and JIA has become better understood, the identity of the target proteins of this modification has remained elusive. In this study, we evaluated serum from patients with various subtypes of JIA to investigate the presence of anti-deiminated (citrullinated) fibrinogen and anti-citrullinated ?-enolase antibodies, and their association with RF and anti-CCP antibody isotypes. Methods Sera were obtained from 96 JIA patients, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. All sera were measured for antibodies against citrullinated and native fibrinogen and ?-enolase by an enzyme linked immunosorbent assay (ELISA). In addition, all sera were assayed for anti-CCP antibody isotypes and rheumatoid factor (RF) isotypes by ELISA. The relationship between anti-citrullinated fibrinogen and anti-?-enolase antibodies and disease activity and joint damage were also investigated. All results were correlated with clinical and laboratory parameters using Spearman's rho correlation coefficient. Multiple logistic regression analysis was utilized to identify which variables were associated with joint erosions and diagnosis of JIA. Results Thirty-one JIA patients (32%) demonstrated reactivity to citrullinated fibrinogen and 9 (9%) to citrullinated ?-enolase. Reactivity to citrullinated fibrinogen and ?-enolase was predominantly found in IgM RF-positive polyarthritis patients. Fourteen JIA patients reacted with native ?-enolase and a higher percentage of SLE patients reacted with citrullinated ?-enolase when compared to JIA patients. Anti-citrullinated fibrinogen antibodies correlated with the presence of IgG anti-CCP antibodies and IgA and IgM RF. The presence of anti-citrullinated ?-enolase antibodies correlated with IgA anti-CCP antibodies. IgG anti-CCP antibodies were significantly associated with joint damage and anti-citrullinated fibrinogen antibodies were strongly associated with JIA when compared to control groups. Anti-citrullinated fibrinogen antibodies demonstrated high sensitivity (81%) for IgM RF-positive polyarticular JIA. IgG anti-CCP antibodies had the highest specificity (95%) for JIA, with anti-citrullinated fibrinogen antibodies, IgA anti-CCP antibodies and IgA RF all following at 84%. Conclusions JIA patient sera exhibited strong reactivity to anti-citrullinated fibrinogen antibodies and demonstrated high sensitivity and specificity for JIA, primarily in IgM RF-positive polyarthritis patients. Fibrinogen is one of several protein targets for citrullination in JIA. PMID:21439056

2011-01-01

301

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

302

Sensitive giant magnetoresistive-based immunoassay for multiplex mycotoxin detection.  

PubMed

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 microm x 90 microm, arranged in an 8 x 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 B(1), 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

2010-03-15

303

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

304

Current Trends in Immunoassay-Based Kits for Pesticide Analysis  

Microsoft Academic Search

Detection of pesticides and their metabolites in food and environmental samples in real time is the goal of many industries. Immunoassay technology has several attributes that make it a useful tool for screening purposes (e.g., selectivity, sensitivity, portability, and rapid turnaround time). Approximately 90% of the developed immunoassays for the pesticide residue analysis use the ELISA technique. Commercial kits are

José Antonio Gabaldón; Angel Maquieira; Rosa Puchades

1999-01-01

305

Enzyme Reactions  

NSDL National Science Digital Library

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

School, Minerva D.

2011-10-03

306

Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays  

SciTech Connect

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

Liu, Guodong; Lin, Yuehe

2007-12-15

307

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

308

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

309

Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays.  

PubMed

A monoclonal antibody (mAb) was produced against immunoglobulin M (IgM) of South American camelids. A single radial immunodiffusion (SRID) assay and a competitive enzyme-linked immunosorbent assay (ELISA) were developed to measure IgM in serum samples. Isotype and specificity of the mAb were assessed. The performance of the SRID assay was preliminarily evaluated in terms of working range, plate stability over a 4-week period, and initial intra- and interassay variation. The concentration of IgM was determined in 55 samples by SRID assay and ELISA, and results were not significantly different by t-test (0.64 ± 0.19 mg/ml for the SRID assay, and 0.58 ± 0.24 mg/ml for ELISA; P = 0.1489). The mAb was shown to be stable over the 4-week evaluation period, and the SRID assay was reproducible when tested in triplicate for intra-assay variability and in quadruplicate for interassay variability, with a percentage coefficient of variation of less than or equal to 5%. Also, the SRID assay proved to be sensitive enough to measure IgM levels in undiluted serum samples, and had a good correlation with ELISA. The current study is intended to submit a preliminary report of a mAb against IgM of South American camelids, and suggest the future potential of the mAb developed for diagnostic application, including use in the SRID assay. PMID:25057160

Friedrich, Adrián; Ledesma, Martín; Landone, Ignacio; Ferrari, Alejandro; Leoni, Juliana

2014-09-01

310

Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay.  

PubMed

Enzyme-linked immunosorbent assay (ELISA) methods based on natural enzyme-labeled probes have been applied in the immunoassays, but most have some inevitable limitations (e.g. harsh preparation, purification and storage) and are unsuitable for routine use. Herein we synthesized a new class of irregular-shaped platinum nanoparticles (ISPtNP) with a mean length of 7.0 nm and a narrowing width from 2.0 to 5.0 nm along the longitudinal axes, which were utilized as peroxidase-like mimics for the development of colorimetric immunoassays. Compared with bioactive horseradish peroxidase (HRP), the synthesized ISPtNP exhibited a low Km value (~0.12 mM) and a high Kcat value (~2.27×10(4)s(-1)) for 3,3',5,5'-tetramethylbenzidine (TMB) with strong thermal stability and pH tolerance. The catalytic mechanism of the ISPtNP toward TMB/H2O2 was for the first time discussed and deliberated in this work. Based on a sandwich-type assay format, two types of colorimetric immunoassay protocols were designed and developed for the detection of rabbit IgG (RIgG, as a model) by using the synthesized ISPtNP and conventional HRP as the labeling of detection antibodies, respectively. Similar detection limits (LODs) of 2.5 ng mL(-1) vs. 1.0 ng mL(-1) were obtained toward RIgG with the ISPtNP labeling compared to HRP format. Intra- and inter-assay coefficients of variation were less than 13%. Importantly, the ISPtNP-based assay system could be suitable for use in a mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity. PMID:23601285

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

2013-05-01

311

Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen?  

PubMed Central

Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population. PMID:21697342

Percival, Ann; Thorkildson, Peter; Kozel, Thomas R.

2011-01-01

312

Detection of IgM antibrucella antibody in the absence of IgGs: a challenge for the clinical interpretation of brucella serology.  

PubMed

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

2014-12-01

313

Detection of IgM and IgG against hepatitis E virus in serum and meat juice samples from pigs at slaughter in Bavaria, Germany.  

PubMed

Hepatitis E virus (HEV) is an emerging foodborne pathogen with domestic and wild pigs (and likely other species such as deer or rabbits) recognized as reservoir. Pathogenesis in pigs usually leads to an asymptomatic course of disease. Since there is no enzyme-linked immunosorbent assay (ELISA) kit for the detection of anti-HEV antibodies in pigs commercially available, the objective of this study was to assess the seroprevalence in fattening pigs at slaughter and at herd level using a newly developed ELISA based on genotype (GT) 1 and GT 3 in Bavaria, Germany. Based on 516 serum and 198 meat juice samples collected from different herds at four different Bavarian slaughterhouses, the overall seroprevalence of anti-HEV IgG in serum and meat juice samples was 68.6% and 67.6%, respectively. Analyzing the serum for the presence of anti-HEV IgM, 36/516 (7%) were positive for anti-HEV IgM. At herd level, most of the herds were seropositive for anti-HEV antibodies. The present study shows that HEV is widespread among the Bavarian pig population and that some pigs might test positive for anti-HEV IgM even at the age of slaughter. Also, meat juice serves as an equivalent matrix to serum to test for anti-HEV antibodies in pigs. PMID:22690762

Wacheck, Silke; Werres, Carolin; Mohn, Ulrich; Dorn, Silvia; Soutschek, Erwin; Fredriksson-Ahomaa, Maria; Märtlbauer, Erwin

2012-07-01

314

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

315

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

316

Automated fluorescence polarization immunoassay for monitoring streptomycin.  

PubMed Central

The fluorescence polarization immunoassay technique has been used previously for the assay of vancomycin and the aminoglycoside antibiotics (gentamicin, tobramycin, and amikacin). We extended this technique to assay streptomycin. Fluorescein-labeled streptomycin was used as the tracer, and antiserum specific for streptomycin was raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence was determined in a specially designed fluorometer (Abbott TDx). Because of the instrument design, the possibility of fluorescent interferences was eliminated. The coefficient of variation within run was 4% (n = 5), and between run it was 5% (n = 5). We compared the fluorescence polarization immunoassay technique (TDx streptomycin) with a conventional bioassay, using Bacillus subtilis for clinical specimens (n = 39). A least-squares linear regression analysis gave a slope of 1.16, an intercept of 2.41 mg/liter, and a correlation coefficient of 0.885. Repeat analyses by both techniques showed that the largest discrepancies could be explained by the imprecision of the bioassay. PMID:6347058

Schwenzer, K S; Anhalt, J P

1983-01-01

317

Fluorimetric Immunoassay for Multianalysis of Aflatoxins  

PubMed Central

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

2013-01-01

318

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

319

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

320

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

321

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

322

IgM nephropathy: timely response to a call for action  

PubMed Central

Immunoglobulin M (IgM) is the largest antibody molecule and is deposited in the glomeruli in a wide variety of both primary and secondary glomerulopathies. The data on its pathogenic role are conflicting till date. A recent study provides evidence for the involvement of natural antibody IgM in fixing and activating complement and causing glomerular injury, proteinuria, and glomerulosclerosis in an animal model. This finding is important in understanding the pathogenesis of the related disorder of IgM nephropathy. PMID:25340155

Mubarak, Muhammed; Nasri, Hamid

2014-01-01

323

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

PubMed Central

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

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

2014-01-01

324

Enzyme Kinetics  

NSDL National Science Digital Library

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

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

1998-01-01

325

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

326

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

327

Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection  

PubMed Central

Background The CombiMatrix ElectraSense® microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs. Methodology/Principal Findings An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense® microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different. Conclusions/Significance Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay. PMID:20333309

Cooper, John; Yazvenko, Nina; Peyvan, Kia; Maurer, Karl; Taitt, Chris R.; Lyon, Wanda; Danley, David L.

2010-01-01

328

Microretroreflector-sedimentation immunoassays for pathogen detection.  

PubMed

Point-of-care detection of pathogens is medically valuable but poses challenging trade-offs between instrument complexity and clinical and analytical sensitivity. Here we introduce a diagnostic platform utilizing lithographically fabricated micron-scale forms of cubic retroreflectors, arguably one of the most optically detectable human artifacts, as reporter labels for use in sensitive immunoassays. We demonstrate the applicability of this novel optical label in a simple assay format in which retroreflector cubes are first mixed with the sample. The cubes are then allowed to settle onto an immuno-capture surface, followed by inversion for gravity-driven removal of nonspecifically bound cubes. Cubes bridged to the capture surface by the analyte are detected using inexpensive, low-numerical aperture optics. For model bacterial and viral pathogens, sensitivity in 10% human serum was found to be 10(4) bacterial cells/mL and 10(4) virus particles/mL, consistent with clinical utility. PMID:25133758

Garvey, Gavin; Shakarisaz, David; Ruiz-Ruiz, Federico; Hagström, Anna E V; Raja, Balakrishnan; Pascente, Carmen; Kar, Archana; Kourentzi, Katerina; Rito-Palomares, Marco; Ruchhoeft, Paul; Willson, Richard C

2014-09-16

329

Evaluation of a dipstick enzyme-linked immunosorbent assay for detection of antibodies to dengue virus.  

PubMed

Accurate serological confirmation of dengue (DEN) infection is difficult, because simple reliable assays for the detection of DEN antibodies are not available. To address this problem, a dipstick enzyme-linked immunosorbent assay (ELISA) was evaluated. The dipstick contained dots of serially diluted DEN 2 antigen. To detect immunoglobulin G (IgG), the dipstick was processed through four reaction cuvettes containing test serum, enhancer, enzyme-conjugated anti-human IgG and IgM antibody, and substrate. Total assay time was 45 min. To detect IgM, the serum was passed through a protein G device to remove IgG. The dipstick was then processed as before, except that the incubation times were longer and enzyme-conjugated anti-human IgM was used. The total assay time was 3 h. The dipstick ELISA results were compared with results from microplate ELISA. The IgG dipstick ELISA showed a sensitivity of 95.2% and a specificity of 100% compared to an IgG microplate ELISA with serum samples from 125 individuals living in an area in which DEN is endemic. In tests with 75 serum samples from patients with clinically suspected acute DEN infections, the IgM dipstick ELISA showed a sensitivity of 97.9% and specificity of 100% compared to those of an IgM antibody capture microplate ELISA. These results showed that the dipstick ELISA was a sensitive and specific test for the detection of either DEN IgM or IgG in human serum. The dipstick ELISA was also shown to be useful for detecting seroconversions to DEN IgM or IgG in paired serum samples from 20 patients with virus isolation-confirmed acute DEN infections. PMID:9220163

Wu, S J; Hanson, B; Paxton, H; Nisalak, A; Vaughn, D W; Rossi, C; Henchal, E A; Porter, K R; Watts, D M; Hayes, C G

1997-07-01

330

Competitive Quenching Fluorescence Immunoassay for Chlorophenols Based on  

E-print Network

and Department of Entomology and UCD Cancer Center, University of California, Davis, California 95616 An improved. The detectability achieved is sufficient for occupational exposure risk assessment. Fluorescent detection methods the opportunity for miniaturization and high- throughput screening. Fluorescence immunoassays (fluoroimmu

Hammock, Bruce D.

331

A computer program for estimating imprecision characteristics of immunoassays.  

PubMed

A reliable numerical algorithm is described, together with a computer program written in FORTRAN IV and FORTRAN 77, for estimating a three-parameter variance function by approximate conditional likelihood. The function is sufficiently flexible to provide for a several thousand-fold relative change in variance and appears to be a good model for the severely heteroscedastic results obtained from immunoassays. The computer program is primarily intended for summarizing imprecision characteristics of immunoassays in the form of imprecision profiles, but the estimated variance functions have additional application whenever further parametric analysis of immunoassay results is undertaken (e.g., as a weighting function when immunoassay results are used in a least-squares regression analysis). The flexibility of the function implies useful application in any area where heteroscedasticity is particularly severe. PMID:2335070

Sadler, W A; Smith, M H

1990-04-01

332

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

333

Probing the Warm Hot IGM with Broad Ly? Absorbers  

NASA Astrophysics Data System (ADS)

Broad Lyman alpha absorbers (BLAs) offer an alternative method to highly-ionized metal lines for tracing the warm-hot intergalactic medium (WHIM) at T>105 K. Such features are thermally broadened to line widths of greater than 40 km s-1 and are typically weak (N<1014 cm-2). Observing such low-contrast spectral features and clearly identifying lines likely shaped by thermal broadening requires high-quality data and accurate continuum placement. Thanks to the high sensitivity of the Cosmic Origins Spectrograph (COS), high signal-to-noise (S/N) far-UV spectra of faint AGN are now attainable in modest observing times. We present early COS G130M/G160M observations (lambda=1130-1800 A) of the quasars PKS 0405-123 (z=0.57, S/N=30-50) and 1ES 1553+113 (z 0.40, S/N=15-20). Both sight lines are rich in IGM absorption systems and the high S/N allows us to catalog several dozen BLA features. We compare the new COS observations of PKS 0405-123 with archival STIS/E140M observations (S/N=10-15) and find qualitative agreement with roughly half of the published BLA systems. As comparable previous observations of 1ES 1553+113 do not exist, we present several new BLA candidates along this sight line. We measure the BLA detection frequency, determine the coincidence of BLAs with other WHIM tracers such as OVI, and estimate the contribution of BLAs to the total baryon density of the universe. Finally, we outline some of the biases and systematic assumptions inherent in BLA WHIM surveys and discuss how each affects the search for missing baryons.

Danforth, Charles; Stocke, J. T.; Shull, J.; Savage, B. D.; Sembach, K. R.

2010-01-01

334

Detection of narcotics with an immunoassay film badge  

SciTech Connect

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

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

1993-12-31

335

Development of immunoassays for human urokinase  

NASA Technical Reports Server (NTRS)

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

Atassi, M. Zouhair

1988-01-01

336

Purification and molecular characterization of IgM in olive barb, Puntius Sarana.  

PubMed

In the present article, immunoglobulin (Ig) of Puntius sarana (a vulnerable medium carp species) was purified by affinity chromatography, characterized, and identified as only IgM type with a native molecular weight of 879 kDa having one heavy (88 kDa) and one light (26 kDa) chain. Further, the developed rabbit antisera against IgM was found to be quite specific to P. sarana IgM and used in ELISA to measure the antibody titer in P. sarana at different time periods, against an antigen (hemocyanin) injection with and without adjuvant. The antibody titer was significantly higher in most of the time periods in both groups, however, the adjuvant-treated group showed higher antibody titer at days 43 and 90, compared to non adjuvant-treated group. Further, the partial IgM sequence was amplified and its expression level was checked during ontogenesis. The IgM transcript was detected from unfertilized egg stage to 4 days post fertilization (dpf) and again reappeared at 21 dpf whereas during infection with Aeromonas hydrophila, significantly marked up-regulation of the gene was observed at 12 hr, 24 hr, and 7 days post-infection time periods indicating the role of IgM during early embryonic time period as well as during bacterial pathogenesis. PMID:24654823

Das, Abhilipsa; Sahoo, Pramoda Kumar; Mohanty, Jyotirmaya; Garnayak, Sushil Kumar

2014-01-01

337

Enzyme Informatics  

PubMed Central

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

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

2012-01-01

338

Circulating Natural IgM Antibodies Against Angiogenin in the Peripheral Blood Sera of Patients with Osteosarcoma as Candidate Biomarkers and Reporters of Tumorigenesis  

PubMed Central

Background: Tumor immunology research has led to the identification of a number of tumor-associated self antigens, suggesting that most tumors trigger an immunogenic response, as is the case in osteosarcoma, where the detection of natural serum IgM antibodies might achieve the diagnosis of osteosarcoma. Natural IgM antibodies to tumor-associated proteins may expand the number of available tumor biomarkers for osteosarcoma and may be used together in a serum profile to enhance test sensitivity and specificity. Natural IgM antibodies can be consistently detected in the peripheral blood sera months to years before the tumor is diagnosed clinically. The study of the level of a potential biomarker many months (or years) prior to diagnosis is fundamentally important. Integrated circulating and imaging markers in clinical practice treating osteosarcoma have potential applications for controlling tumor angiogenesis. Objectives: To study the expression of natural IgM antibodies to the tumor antigens of angiogenesis in the peripheral blood sera of osteosarcoma patients and healthy individuals, and to develop serum-based predictive biomarkers. Methods: Peripheral venous blood samples were collected from 117 osteosarcoma patients and 117 patients with other tumors. All diagnosis was histologically confirmed. Staging of patients was performed according to the Enneking Surgical Staging System. The control group consisted of 117 age- and sex- matched healthy individuals. In this study, novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive enzyme-linked immunosorbent assay (ELISA) method to detect angiogenin (ANG)–IgM directly in the peripheral blood sera of humans. Results: Serum ANG–IgM levels are significantly higher in osteosarcoma patients than in healthy individuals (P < 0.005). Serum ANG–IgM levels varied widely, but were highly dependent on the concentration of IgM (r = 0.85; P < 0.0005). We found ANG–IgM in the sera of 85% of newly diagnosed osteosarcoma patients and ANG–IgM levels were significantly higher in osteosarcoma patients compared to any other tumors (P < 0.001). Conclusions: These results demonstrated that the combined biomarker ANG–IgM has greater sensitivity and specificity in early diagnosis of osteosarcoma patients than the traditional biomarkers (ANG and vascular endothelial growth factor). Circulating ANG–IgM immune complexes can potentially serve as a biomarker for increased risk of osteosarcoma, because relatively high serum levels were also detected in otherwise healthy individuals with a first degree family history of osteosarcoma and in patients with a diagnosis of benign conditions. Immunological aspects of angiogenesis for managing osteosarcoma will have a practical value in early diagnosis, prognosis and monitoring response to antiangiogenic therapy. PMID:24179386

Savitskaya, Yulia A.; Rico, Genaro; Linares, Luis; González, Roberto; Téllez, René; Estrada, Eréndira; Marín, Norma; Martínez, Elisa; Alfaro, Alfonso; Ibarra, Clemente

2010-01-01

339

Serodiagnosis of typhoid fever by enzyme-linked immunosorbent assay determination of anti-Salmonella typhi lipopolysaccharide antibodies.  

PubMed Central

Serum samples from 22 patients with proven typhoid fever, 60 febrile nontyphoidal patients, and 120 healthy subjects were tested for immunoglobulin A (IgA), IgG, and IgM anti-Salmonella typhi lipopolysaccharide antibodies by an enzyme-linked immunosorbent assay. The levels of all three classes of immunoglobulin anti-lipopolysaccharide were higher in typhoid patients than in controls; the test for IgM anti-lipopolysaccharide gave the best discrimination between typhoid and nontyphoidal sera. The absorbance values obtained with the enzyme-linked immunosorbent assay for IgM anti-lipopolysaccharide were highly correlated to the titers of anti-O agglutinins. However, the enzyme-linked immunosorbent assay was much more specific than the Widal test, and hence it could be a useful tool for the serological diagnosis of typhoid fever with a single blood sample. PMID:6386877

Nardiello, S; Pizzella, T; Russo, M; Galanti, B

1984-01-01

340

Natural IgM Is Produced by CD5- Plasma Cells That Occupy a Distinct Survival Niche in Bone Marrow.  

PubMed

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

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

2015-01-01

341

Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay  

Microsoft Academic Search

Brain-derived neurotrophic factor (BDNF) is one of several endogenous proteins that play key roles in neuronal development and homeostasis. We describe here the characterization and use of a sensitive and specific enzyme-linked immunoassay (EIA) for BDNF protein. Recombinant BDNF was detected at concentrations as low as 10 pg\\/ml, whereas the EIA did not detect NT-3, NT-4\\/5, or NGF at concentrations

Susan F. Radka; Patricia A. Hoist; Michelle Fritsche; C. Anthony Altar

1996-01-01

342

A multi-allergen standard for calibration of immunoassays: CREATE principles applied to eight purified allergens  

PubMed Central

Background Allergen measurements are widely used for environmental exposure assessments and for determining the potency of allergen vaccines, yet few purified allergen standards have been developed. The aim of the study was to develop a single standard containing multiple purified allergens that could be used in enzyme immunoassays and in multiplex arrays for standardization of allergen measurements. Methods Eight purified allergens were formulated into a single multi-allergen, or “universal”, standard based on amino acid analysis. Dose response curves were compared with previous individual ELISA standards and allergen measurements of house dust extracts to obtain correction factors. Measured allergen concentrations were also modeled using linear regression and the predictive accuracy was determined. Results Parallel dose response curves were obtained between the universal allergen standard and the individual ELISA standards, with close agreement between curves for 5/8 allergens. Quantitative differences of >2-fold were observed for Fel d 1, Can f 1 and Der f 1, which were confirmed by analysis of house dust extracts. Correction factors were developed that allowed ELISA data to be expressed in terms of the universal standard. Linear regression data confirmed the predictive accuracy of the universal standard. Conclusion This study shows that a single standard of eight purified allergens can be used to compare allergen measurements by immunoassay. This approach will improve continuity of environmental exposure assessments and provide improved standardization of allergy diagnostics and vaccines used for immunotherapy. PMID:22092159

Filep, S; Tsay, A.; Vailes, L.; Gadermaier, G.; Ferreira, F.; Matsui, E.; King, E.M.; Chapman, M. D.

2011-01-01

343

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

PubMed Central

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

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

2012-01-01

344

Surface-enhanced Raman scattering for immunoassay based on the biocatalytic production of silver nanoparticles.  

PubMed

We have reported on a novel enzyme immunoassay method for the detection of protein using biocatalytic silver nanoparticles as an enhanced substrate based on surface-enhanced Raman scattering (SERS). First, ascorbic acid was converted from ascorbic acid 2-phosphate by alkaline phosphatase immobilized on polystyrene microwells after a typical sandwich immunoreaction. Then Ag(I) ions were reduced to silver nanoparticles by the obtained ascorbic acid, which would result in a SERS signal when Raman dyes were absorbed. Using human IgG as a model protein, a wide linear dynamic range (1 to 100 ng ml(-1)) was reached with a low detection limit (0.02 ng ml(-1)) under the optimized assay conditions. Moreover, the production of an enhanced substrate was chosen as the signaling element in this method, which demonstrates a new way for SERS-based quantitative detection. These results suggest that the application of SERS enhanced by biocatalytic production of metal nanopaticles holds a promising potential for a sensitive immunoassay. PMID:19276589

Chen, Jiwei; Luo, Yan; Liang, Yi; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2009-03-01

345

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.  

PubMed

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods. PMID:25421624

Shan, Wen C; Cui, Ya L; He, Xin; Zhang, Lei; Liu, Jing; Wang, Jian P

2015-01-01

346

A secondary antibody format chemiluminescence immunoassay for the determination of estradiol in human serum.  

PubMed

A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl(3)COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL(-1). The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum. PMID:20801358

Xin, Tian-Bing; Chen, Hui; Lin, Zhen; Liang, Shu-Xuan; Lin, Jin-Ming

2010-09-15

347

AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level  

NASA Astrophysics Data System (ADS)

AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

Smith, David F.; Stults, Nancy L.

1996-04-01

348

Treatment Failure Related to Intrathecal Immunoglobulin M (IgM) Synthesis, Cerebrospinal Fluid IgM, and Interleukin-10 in Patients with Hemolymphatic-Stage Sleeping Sickness?  

PubMed Central

Human African trypanosomiasis treatment is stage dependent, but the tests used for staging are controversial. Central nervous system involvement and its relationship with suramin treatment failure were assessed in 60 patients with parasitologically confirmed hemolymphatic-stage Trypanosoma brucei gambiense infection (white blood cell count of ?5/?l and no trypanosomes in the cerebrospinal fluid [CSF]). The prognostic value of CSF interleukin-10, immunoglobulin M (IgM; as determined by nephelometry and the point-of-care LATEX/IgM test), total protein, and trypanosome-specific antibody was assessed. The IgM and interleukin-10 levels in serum were measured; and the presence of neurological signs, intrathecal IgM synthesis, and blood-CSF barrier dysfunction was determined. After suramin treatment, 14 of 60 patients had relapses (23%). Relapses were significantly correlated with intrathecal IgM synthesis (odds ratio [OR], 46; 95% confidence interval [CI], 8 to 260), a CSF IgM concentration of ?1.9 mg/liter (OR, 11.7; 95% CI, 2.7 to 50), a CSF end titer by the LATEX/IgM assay of ?2 (OR, 10.4; 95% CI, 2.5 to 44), and a CSF interleukin-10 concentration of >10 pg/ml (OR, 5; 95% CI, 1.3 to 20). The sensitivities of these markers for treatment failure ranged from 43 to 79%, and the specificities ranged from 74 to 93%. The results show that T. brucei gambiense-infected patients who have signs of neuroinflammation in CSF and who are treated with drugs recommended for use at the hemolymphatic stage are at risk of treatment failure. This highlights the need for the development and the evaluation of accurate point-of-care tests for the staging of human African trypanosomiasis. PMID:17428948

Lejon, Veerle; Robays, Jo; N'Siesi, François Xavier; Mumba, Dieudonné; Hoogstoel, Annemie; Bisser, Sylvie; Reiber, Hansotto; Boelaert, Marleen; Büscher, Philippe

2007-01-01

349

Nanogold-polyaniline-nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone.  

PubMed

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold-polyaniline-nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab(2)) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0mmolL(-1) H(2)O(2) by using GPG-labeled HRP-Ab(2) as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab(2). Several labeling protocols comprising HRP-Ab(2), nanogold particle-labeled HRP-Ab(2), and polyaniline nanospheres-labeled HRP-Ab(2), were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab(2). With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab(2) to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20?IUmL(-1) with a detection limit (LOD) of 0.005?IUmL(-1) TSH at the 3s(B) criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH. PMID:22790703

Cui, Yuling; Chen, Huafeng; Hou, Li; Zhang, Bing; Liu, Bingqian; Chen, Guonan; Tang, Dianping

2012-08-13

350

Sequential injection immunoassay utilizing immunomagnetic beads.  

PubMed

A novel sequential injection immunoassay (SIIA) method is described which utilizes immunomagnetic beads to investigate short-time antibody binding. The method is versatile and flexible and may therefore be adapted to many different applications. Initial results for a competitive assay are also presented. The immunomagnetic bead reactor is created within the flowing stream by retaining immunomagnetic beads with an electromagnet to form an open tube reactor. Thus, the spent beads may be discharged after each analysis. This eliminates the problems of instability of reaction surfaces and eliminates the need for additional time traditionally required for regeneration of the solid-reacting phase in order to not only save time and increase sampling frequency but also to provide each individual sampling cycle with a fresh, uniform portion of beads. The spent beads are collected off line and may be regenerated later. Short-time binding kinetic studies demonstrate linear initial binding under 1 min, which then begins to reach saturation in approximately 10 min. Competitive binding assays of monoclonal mouse IgG (MRC OX-19) to polyclonal sheep anti-mouse IgG immobilized to the immunomagnetic beads show reproducible linear displacement in 30-120-s reactions. Fluorescence detection is utilized with a detection limit of 155 ng/mL, and since the reaction time is typically 2 min or shorter, the sampling frequency is 30 samples/h. PMID:1503215

Pollema, C H; Ruzicka, J; Christian, G D; Lernmark, A

1992-07-01

351

Single molecule immunoassay on plasmonic platforms.  

PubMed

We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-665 on a plasmonic platform of self- assembled colloidal structures (SACS) of silver prepared on a semitransparent silver film. A SeTau-665 immunoassay was performed on this platform and a control glass slide. The fluorescence properties of this label substantially change due to plasmonic interactions. While the average brightness increase of SeTau 665 in ensemble measurements was about 70-fold, fluorescence enhancements up to four-hundred times were observed on certain "hot spots" for single molecule measurements. The intensity increase is strongly correlated with a simultaneous decrease in fluorescence lifetime in these "hot spots". The large increase in brightness allows the reduction of the excitation power resulting in a reduced background and increased photostability. The remarkable fluorescence enhancements observed for SeTau 665 on our plasmonic platform should allow to substantially improve single molecule detection and to reduce the detection limits in sensing devices. PMID:19929821

Luchowski, R; Matveeva, E G; Shtoyko, T; Sarkar, P; Patsenker, L D; Klochko, O P; Terpetschnig, E A; Borejdo, J; Akopova, I; Gryczynski, Z; Gryczynski, I

2010-01-01

352

Multiplex lateral flow immunoassay for mycotoxin determination.  

PubMed

A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 ?g/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 ?g/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 ?g/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins. PMID:24745689

Song, Suquan; Liu, Na; Zhao, Zhiyong; Njumbe Ediage, Emmanuel; Wu, Songling; Sun, Changpo; De Saeger, Sarah; Wu, Aibo

2014-05-20

353

Concentration Gradient Immunoassay I. A Rapid Immunoassay Based on Interdiffusion and Surface Binding in a Microchannel  

PubMed Central

We describe a novel microfluidic immunoassay method based on the diffusion of a small molecule analyte into a parallel-flowing stream containing cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch et al. Nature Biotechnology,19:461?465 (2001)), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analog of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/sec, the assays are complete within 10 minutes. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low-molecular weight analytes for point-of-care diagnostic instrumentation. PMID:17437332

Nelson, Kjell E.; Foley, Jennifer O.; Yager, Paul

2008-01-01

354

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

PubMed Central

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

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

2014-01-01

355

Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens.  

PubMed

In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 ?g L(-1) to as low as 0.07 ?g L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. PMID:24745749

Wang, Zhanhui; Zhang, Huiyan; Ni, Hengjia; Zhang, Suxia; Shen, Jianzhong

2014-04-11

356

A new highly sensitive immunoassay for cytokines by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).  

PubMed

Non-isotopic immunoassays for human tumor necrosis factor alpha (TNF alpha) and human interleukin-6 (IL-6) were established by employing the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) system based on the time-resolved fluoroimmunoassay technique with europium-labeled antibody. Compared to enzyme-linked immunosorbent assays and bioassays, the sensitivity and range of measurement were significantly increased by applying the DELFIA systems to TNF alpha and IL-6. TNF alpha was measurable from 100 fg/ml to 10 ng/ml with the TNF alpha-DELFIA and IL-6 was measurable from 100 fg/ml to 1 ng/ml with the IL-6-DELFIA. PMID:1564324

Ogata, A; Tagoh, H; Lee, T; Kuritani, T; Takahara, Y; Shimamura, T; Ikegami, H; Kurimoto, M; Yoshizaki, K; Kishimoto, T

1992-04-01

357

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies.  

PubMed

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD. PMID:25525116

Tsiantoulas, Dimitrios; Perkmann, Thomas; Afonyushkin, Taras; Mangold, Andreas; Prohaska, Thomas A; Papac-Milicevic, Nikolina; Millischer, Vincent; Bartel, Caroline; Hörkkö, Sohvi; Boulanger, Chantal M; Tsimikas, Sotirios; Fischer, Michael B; Witztum, Joseph L; Lang, Irene M; Binder, Christoph J

2015-02-01

358

Influenza virus-specific neutralizing IgM antibodies persist for a lifetime.  

PubMed

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

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

2014-11-01

359

Serum IgM concentrations in normal, fit horses and horses with lymphoma or other medical conditions.  

PubMed

The purposes of this study were to (1) prospectively establish serum IgM and IgG concentrations in normal, fit, adult horses over time and (2) determine the accuracy of serum IgM concentrations for diagnosing lymphoma. Serial IgM and IgG concentrations were measured with a radial immunodiffusion assay in 25 regularly exercised horses at 6-week intervals. Horses had serum IgM concentrations ranging from 50 to 242 mg/dL over 5 months, with 20% of horses having IgM < or = 60 mg/dL. The normal range for IgM in fit horses should be considered 103 +/- 40 mg/dL and a cut-point for an IgM deficiency, < or = 23 mg/dL. IgG concentrations ranged from 1,372 to 3,032 mg/dL. Retrospectively, medical records of adult horses (n = 103) admitted to the Cornell University Hospital for Animals for which serum IgM was measured were examined. Horses were categorized as "lymphoma negative" (n = 34) or "lymphoma positive" (n = 18). The sensitivity and specificity of a serum IgM concentration (< or = 60 mg/dL) for detecting equine lymphoma was 50 and 35%, respectively. At the new cut-point (< or = 23 mg/dL), the sensitivity was low at 28% and the specificity improved to 88%. The negative predictive values at various population prevalences indicate that a horse with a high serum IgM (> 23 mg/dL) is unlikely to have lymphoma, whereas the positive predictive value (70%) does not allow for reliable determination of lymphoma in a horse with serum IgM < or = 23 mg/dL. Therefore, serum IgM concentrations should not be used as a screening test for equine lymphoma. PMID:12774976

Perkins, G A; Nydam, D V; Flaminio, M J B F; Ainsworth, D M

2003-01-01

360

The effect of cortisol administration on blood plasma immunoglobulin M (IgM) concentrations in masu salmon ( Oncorhynchus masou )  

Microsoft Academic Search

Immunoglobulin M (IgM) is known as a main factor in the humoral immune system of teleosts. In the present study, the effect\\u000a of cortisol on plasma IgM concentrations was investigated using a specific antibody to IgM in masu salmon (Oncorhynchus masou). Cortisol was orally administered each day for 2 weeks at a dose of 1 mg g?1 in the diet,

Masaki Nagael; Hirotoshi Fuda; Kazuhiro Ura; Hiroshi Kawamura; Shinji Adachi; Akihiko Hara; Kohei Yamauchil

1994-01-01

361

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

362

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

E-print Network

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild for vitellogenin purification and samples from adult males were used for comparison. No differences were detected

Ray, David

363

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

EPA Science Inventory

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

364

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

EPA Science Inventory

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

365

Synthesis of Haptens and Protein Conjugates for the Development of Immunoassays for the Insect Growth Regulator  

E-print Network

Synthesis of Haptens and Protein Conjugates for the Development of Immunoassays for the Insect design; synthesis of haptens and protein conjugates; polyclonal antibodies; ELISA; immunoassay to produce protein conjugates and rabbit polyclonal antisera. Amino derivatives of fenoxycarb at the terminal

Hammock, Bruce D.

366

An electrochemical amplification immunoassay using bi-electrode signal transduction system.  

PubMed

An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity. PMID:19071559

Chen, Zhao-Peng; Jiang, Jian-Hui; Zhang, Xiao-Bing; Shen, Guo-Li; Yu, Ru-Qin

2007-03-30

367

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

PubMed

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

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

2013-01-01

368

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

369

Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.  

PubMed

Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references). PMID:23740388

Wei, Hui; Wang, Erkang

2013-07-21

370

The purification and characterisation of cervine IgM and IgG.  

PubMed

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

Hibma, M H; Griffin, J F

1990-12-01

371

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

PubMed

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

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

2014-12-14

372

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

PubMed Central

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

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

2014-01-01

373

Importance of specific IgM antibodies in 116 patients with various stages of syphilis.  

PubMed Central

We tested 222 serum samples obtained from 51 patients presenting with syphilis, before and after treatment; 117 from 65 patients with a history of syphilis (114) or yaws (3); 77 from 71 patients with no evidence of syphilis; and 1117 serologically negative serum samples. Our tests included the IgM fluorescent treponemal antibody absorbed (IgM-FTA-ABS) and solid phase haemadsorption assay (SPHA) techniques. According to the stage of development of syphilis, IgM antibodies were found in 83-100% of the serum samples. This permitted a precise diagnosis to be made and cure assessed. As IgM antibodies were absent in serum from patients with healed syphilis, resolved syphilis could be distinguished from developing syphilis. The sensitivity (92%) of the IgM-FTA-ABS test was comparable with that of the SPHA (96%), but the SPHA was more specific (97.4%) than the IgM-FTA-ABS test (89.61%). PMID:3884486

Merlin, S; Andre, J; Alacoque, B; Paris-Hamelin, A

1985-01-01

374

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

SciTech Connect

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

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

1986-03-21

375

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

PubMed Central

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

Vojdani, Aristo

2009-01-01

376

An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum.  

PubMed

Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical-chemical-chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH3)6(3+), enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH3)6(3+) is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [?-galactosidase, 4-amino-1-naphthyl ?-D-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V vs. Ag/AgCl) minimizes the interference effect of electroactive species (L-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL(-1) for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples. PMID:24482801

Akanda, Md Rajibul; Joung, Hyou-Arm; Tamilavan, Vellaiappillai; Park, Seonhwa; Kim, Sinyoung; Hyun, Myung Ho; Kim, Min-Gon; Yang, Haesik

2014-03-21

377

Antigen excess in modern immunoassays: to anticipate on the unexpected.  

PubMed

Immunoassays measuring sera with high analyte concentration may be prone to an artifact that causes underestimation of the analyte concentration. This phenomenon is generally described as antigen excess or the prozone effect. Characteristically, serum with high concentrations of a certain analyte can give a false negative/low result when tested at the recommended dilution, but reacts strongly positive upon further dilution. Increased insight of the antigen excess mechanisms and tools to prevent it has reduced the analytical problems caused by prozone effects in daily laboratory practice. However, misinterpretation of laboratory results caused by antigen excess does still occur, in virtually any type of immunoassay. Awareness by the laboratory specialist of the mechanisms underlying antigen excess in the different immunoassays, strategies to detect it, and adequate communication with clinicians can help to avoid reporting false negative test-results. PMID:25461469

Jacobs, Joannes F M; van der Molen, Renate G; Bossuyt, Xavier; Damoiseaux, Jan

2015-02-01

378

Enzyme Reactions  

NSDL National Science Digital Library

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

Maryland Virtual High School

379

Enzyme Action  

NSDL National Science Digital Library

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

Crumlish, Jane

2009-01-01

380

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

PubMed Central

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

2014-01-01

381

Peroxidase-like activity of mesoporous silica encapsulated Pt nanoparticle and its application in colorimetric immunoassay.  

PubMed

Nanomaterial-based artificial enzymes have received great attention in recent year due to their potential application in immunoassay techniques. However, such potential is usually limited by poor dispersion stability or low catalytic activity induced by the capping agent essentially required in the synthesis. In an attempt to address these challenges, here, we studied the novel Pt nanoparticles (NPs) based peroxidase-like mimic by encapsulating Pt NP in mesoporous silica (Pt@mSiO2 NPs). Compared with other nanomaterial-based artificial enzymes, the obtained Pt@mSiO2 NPs not only exhibit high peroxidase-like activity but also have good dispersion stability in buffer saline solution when grafted with spacer PEG. Results show that when the thickness of silica shell is about 9nm the resulting Pt@mSiO2 NPs exhibit the catalytic activity similar to that of Pt NPs, which is approximately 26 times higher than that of Fe3O4 NPs (in terms of Kcat for H2O2). Due to the protection of silica shell, the subsequent surface modification with antibody has little effect on their catalytic activity. The analytical performance of this system in detecting hCG shows that after 5min incubation the limit of detection can reach 10ngmL(-1) and dynamic linear working range is 5-200ngmL(-1). Our findings pave the way for design and development of novel artificial enzyme labeling. PMID:25682428

Wang, Zhifei; Yang, Xia; Yang, Jingjing; Jiang, Yanyun; He, Nongyue

2015-03-01

382

Robust detection of peak signals for lateral flow immunoassays  

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

383

Comparison of 3 automated immunoassays for detection of anti-hepatitis A virus immunoglobulin M in a tertiary care hospital.  

PubMed

Three automated immunoassay kits for anti-Hepatitis A Virus (HAV) IgM-Architect, (Abbott Laboratories, USA), Elecsys (Roche Diagnostics, Germany), and ADVIA Centaur (Siemens Healthcare Diagnostics Inc., USA)-were compared. We included 178 consecutive samples, for which an anti-HAV IgM test was requested at Seoul National University Hospital from September 2009 to January 2010. Reviewing of medical records, reverse transcription (RT)-PCR for HAV RNA, or total anti-HAV assay were performed on 16 (9.0%) samples with discrepant results. The percent agreements (kappas) of the Architect and ADVIA Centaur, Architect and Elecsys, and ADVIA Centaur and Elecsys kits were 96.6% (0.91), 96.6% (0.92), and 97.8% (0.94), respectively. Eight out of 16 discrepant samples showed gray-zone values in Architect but were nonreactive in the others. Slightly earlier seroconversion was suspected in Elecsys. The 3 assays showed comparable performances with excellent agreements in a tertiary care hospital setting. PMID:23483019

Park, Hyewon; Lee, Yu-joo; Seong, Moon-Woo; Lee, Do-Hoon; Park, Myoung Hee; Song, Eun Young

2013-03-01

384

Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum.  

PubMed

Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7-carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay. PMID:21113840

Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Bhanot, Sana; Singh, Rita; Kumar, Dinesh

2010-01-01

385

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

E-print Network

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

Fan, Jianqing

386

Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis  

PubMed Central

Successful treatment of leptospirosis is heavily dependent on early diagnosis and prompt initiation of antibiotic therapy. An ELISA test to detect specific IgM antibodies against LipL32 for early diagnosis of leptospirosis is described and evaluated here. One thousand one hundred and eighty sera from clinically suspected leptospirosis cases were enrolled together with 109 healthy volunteers selected from an endemic area between October 2007 and January 2010. Patients were categorized based on their clinical signs and symptoms. Sera were screened for leptospiral antibodies by the microscopic agglutination test (MAT) using a panel of locally circulating serovars followed by enzyme-linked immunosorbent assay (ELISA) based on recombinant LipL32 from Leptospira interrogans serovar Autumnalis strain N2. The sensitivity and specificity of the ELISA test were determined to establish its diagnostic efficiency. The cut-off value was determined to be 0.205. Overall sensitivity and specificity compared to the MAT were found to be 96.4 and 90.4%, respectively. The LipL32-specific IgM ELISA had good sensitivity and acceptable specificity and may be a candidate for the early serodiagnosis of human leptospirosis. PMID:23683367

Vedhagiri, Kumaresan; Velineni, Sridhar; Timoney, John F; Shanmughapriya, Santhanam; Vijayachari, Paluru; Narayanan, Ramasamy; Natarajaseenivasan, Kalimuthusamy

2013-01-01

387

Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire  

E-print Network

Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring cell subsets, that blood IgM IgD CD27 cells correspond to circulating splenic marginal zone B cells and Cell Biology10 University of Marburg, DE G n tique Humaine des Maladies Infectieuses11 é é INSERM : U

Boyer, Edmond

388

Immunobiology Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring  

E-print Network

1 Immunobiology Human blood IgM "memory" B cells are circulating splenic marginal zone B cells expression profiling of the different blood and splenic B cell subsets, that blood IgM+ IgD+ CD27+ cells cells in human Sandra Weller1 , Moritz C. Braun1 , Bruce K. Tan2 , Andreas Rosenwald2 , Corinne Cordier3

Paris-Sud XI, Université de

389

Enzyme Kinetics  

NSDL National Science Digital Library

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

Krause

2008-10-31

390

Baryonic Content in the Warm-Hot IGM at Low Redshift  

NASA Technical Reports Server (NTRS)

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

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

2007-01-01

391

EUV and FUV Absorption by the Highly Ionized IGM: Physical Conditions and Baryonic Content  

NASA Astrophysics Data System (ADS)

A recent FUSE snapshot observation reveals that HE,0153--4520 z_ QSO 0.451) is the third brightest moderate redshift (z > 0.4) QSO at FUV wavelengths. The line of sight to HE,0153--4520 provides an outstanding opportunity to obtain information on EUV absorption lines formed in the highly ionized IGM. Our proposed 250 ksec integration will build on our recent successful detection of Ne VIII absorption in a multi-phase system at z 0.20701 toward HE,0226--4110 (z_ QSO 0.497). The observations of HE,0153--4520 will allow access to EUV lines of O II, O III, O IV, N IV, and Ne VIII in addition to FUV lines of H I, C II, C III, N II, N III, O I, and O VI. Access to a range of ion states is crucial for determining the origin(s) of the ionization and the baryonic content of the highly ionized IGM. Studies of the physical conditions and ionization of IGM absorption-line systems are an essential step for observational cosmology to make progress in understanding whether the highly ionized IGM is a major reservoir of baryons in the low redshift universe. There are only a few QSOs with z > 0.4 for which a study as detailed as the one proposed here can be conducted.

Savage, B.

392

RESEARCH ARTICLE Open Access Cross-reactivities between human IgMs and the  

E-print Network

and an immunogen in vaccine candidates. Keywords: Cross-reactivity, Dengue Virus, Discrimination, Flavivirus, HumanRESEARCH ARTICLE Open Access Cross-reactivities between human IgMs and the four serotypes of dengue,2 , Philippe Dussart3 , Laetitia Bremand3 and Hugues Bedouelle1,2* Abstract Background: Dengue fever

Paris-Sud XI, Université de

393

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

PubMed Central

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

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

1986-01-01

394

AN EVALUATION OF FIVE COMMERCIAL IMMUNOASSAY DATA ANALYSIS SOFTWARE SYSTEMS  

EPA Science Inventory

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

395

EVALUATION OF FIVE COMMERCIAL IMMUNOASSAY DATA ANALYSIS SOFTWARE SYSTEMS  

EPA Science Inventory

The U.S. EPA, Office of Research and Development, conducted an evaluation of five commercial software systems used for immunoassay data analysis. he evaluation revealed numerous deficiencies. often, the utility of statistical output was compromised by poor documentation. everal d...

396

Alternating current electrokinetics enhanced in situ capacitive immunoassay.  

PubMed

A rapid in situ capacitive immunoassay is presented herein. Conventional immunoassay typically relies on diffusion for transport of analytes in many cases causing long detection time and lack of sensitivity. By integrating alternating current electrokinetics (ACEK) and impedance sensing, this work provides a rapid in situ capacitive affinity biosensing. ACEK induces both fluid flow and particle motion, conveying target molecules toward electrodes immobilized with probes, resulting in rapid enrichment of target molecules and a capacitance change at the ''electrode-fluid'' interface. The benefit of ACEK enhanced immunoassay was demonstrated using the antigen and antibody from Johne's disease (JD) as an example. To clarify the importance of DEP and ACET effects for binding reaction, two different electrode pattern designs for capacitive immunoassay are studied. The asymmetric array and symmetric electrodes exhibit very similar response at lower electric field due to DEP effects, while asymmetric array has remarkable higher response at high-electric field because the convection becomes more important at high field. The disease positive and negative serum samples are distinguished in few minutes. PMID:25258204

Li, Shanshan; Ren, Yukun; Cui, Haochen; Yuan, Quan; Wu, Jie; Eda, Shigetoshi; Jiang, Hongyuan

2014-09-26

397

Monitoring Pesticides and Personal Care Chemicals in Water by Immunoassay  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

398

Microfluidic immunoassay for rapid detection of cotinine in saliva.  

PubMed

A microfluidic immunoassay is successfully developed for rapid analysis of cotinine saliva samples, which is a metabolite of nicotine and is widely used as a biomarker to evaluate the smoking status and exposure to tobacco smoke. The core microfluidic chip is fabricated by polydimethylsiloxane (PDMS) with standard soft lithography. Each chip is capable of eight parallel analyses of cotinine samples. The analyses can be completed within 40 min with 12 ?l sample consumption. The linear detection range is 1?~?250 ng/ml and the minimum detectable concentration is 1 ng/ml respectively. The correlation coefficient of the calibration curve established from standard samples is 0.9989. The immunoassay was also validated by real saliva samples, and the results showed good reproducibility and accuracy. All the results were confirmed with traditional ELISA measurements. The result from microfluidic chip device and ELISA kits showed good correspondence, and the correlation coefficients are higher than 0.99. Compared with traditional technique, this microfluidic immunoassay is more economic, rapid, simple and sensitive, perfect for on-site cotinine measurements as well as for the evaluation of the exposure to tobacco smoking. Moreover, this immunoassay has potential to be applied in the analysis of other biomarkers in human saliva samples. PMID:23832621

Cheng, Kaiping; Zhao, Wang; Liu, Sixiu; Sui, Guodong

2013-12-01

399

A Compact Immunoassay Platform Based on a Multicapillary Glass Plate  

PubMed Central

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

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

2014-01-01

400

AUTOMATED FLOW FLUORESCENT IMMUNOASSAY FOR THE INSECTICIDE THIAMETHOXAM.  

Technology Transfer Automated Retrieval System (TEKTRAN)

A sensitive automated flow fluorescent immunoassay was developed with KinExATM 3000 system for quantitative analysis of the neonicotinoid insecticide thiamethoxam, 3-(2-chlorothiaxol-5-ylmethyl)-5-methyl-4-nitroimino-1,3,5-oxadiazinane. A capillary flow cell contains antigen (thiamethoxam hapten-B...

401

CAPILLARY ELECTROPHORESIS IMMUNOASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID  

EPA Science Inventory

A capillary electrophoresis (CE) immunoassay format for 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated. A fluorescent labeled 2,4-D analog competes with the analyte of interest for a finite number of binding sites provided by anti-2,4-D monoclonal antibodies. CE then pr...

402

DEVELOPMENT OF SENSITIVE MAGNETIC PARTICLE IMMUNOASSAY FOR POLYBROMINATED DIPHENYL ETHERS.  

Technology Transfer Automated Retrieval System (TEKTRAN)

A sensitive magnetic particle based immunoassay for polybrominated diphenyl ether (PBDE) was developed. Rabbit antiserum was produced by immunizing the rabbit with 4-(2,4-dibromo-5-(2,4-dibromophenoxy)phenoxy)butyrate-BSA. The PBDE ligand and horse radish peroxidase were conjugated via NHS and EDA...

403

Multiplexed immunoassay based on micromotors and microscale tags.  

PubMed

This work reports on the coupling of antibody-functionalized micromotors and microwire-tagged proteins for rapid and multiplexed immunoassays. While micromotor-induced mixing accelerates the immunoreaction, tagging the proteins with microscopic particles of different sizes and shapes allows for their multiplexed discrimination, alerting of the presence of a biological threat. PMID:25017813

Vilela, D; Orozco, J; Cheng, G; Sattayasamitsathit, S; Galarnyk, M; Kan, C; Wang, J; Escarpa, A

2014-09-21

404

Ultra-sensitive immunoassay using VCSEL detection system  

E-print Network

proteins and can detect an antigen concentration as low as 1 pg=ml (6.7 femtoMolar). Introduction: Protein and the two detectors are used to track this wavelength shift as materials are being deposited onto the GMR. In this Letter, we report the use of this system in immunoassay with a record high sensitivity to antigen

Cunningham, Brian

405

Placebo-controlled trial of rituximab in IgM anti-myelin–associated glycoprotein neuropathy  

PubMed Central

Objective: To determine whether rituximab 375 mg/m2 was efficacious in patients with immunoglobulin M (IgM) anti-myelin–associated glycoprotein antibody demyelinating neuropathy (IgM anti-MAG demyelinating neuropathy). Methods: Fifty-four patients with IgM anti-MAG demyelinating neuropathy were enrolled in this randomized, double-blind, placebo-controlled trial. The inclusion criteria were inflammatory neuropathy cause and treatment (INCAT) sensory score (ISS) ?4 and visual analog pain scale >4 or ataxia score ?2. The primary outcome was mean change in ISS at 12 months. Results: Twenty-six patients were randomized to a group receiving 4 weekly infusions of 375 mg/m2 rituximab, and 28 patients to placebo. Intention-to-treat analysis, with imputation of missing ISS values by the last observation carried forward method, showed a lack of mean change in ISS at 12 months, 1.0 ± 2.7 in the rituximab group, and 1.0 ± 2.8 in the placebo group. However, changes were observed, in per protocol analysis at 12 months, for the number of patients with an improvement of at least 2 points in the INCAT disability scale (p = 0.027), the self-evaluation scale (p = 0.016), and 2 subscores of the Short Form–36 questionnaire. Conclusions: Although primary outcome measures provide no evidence to support the use of rituximab in IgM anti-MAG demyelinating neuropathy, there were improvements in several secondary outcomes in per protocol analysis. Level of evidence: This study provides Class I evidence that rituximab is ineffective in improving ISS in patients with IgM anti-MAG demyelinating neuropathy. PMID:23667063

Viala, Karine; Nicolas, Guillaume; Créange, Alain; Vallat, Jean-Michel; Pouget, Jean; Clavelou, Pierre; Vial, Christophe; Steck, Andreas; Musset, Lucile; Marin, Benoit

2013-01-01

406

Food Enzymes  

ERIC Educational Resources Information Center

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

McBroom, Rachel; Oliver-Hoyo, Maria T.

2007-01-01

407

Engineering enzymes.  

PubMed

Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of structural and energetic engineering tolerances of the mechanism. Significant barriers to achieving an engineering understanding of enzyme mechanisms arise from natural protein complexity. In certain cases we can surmount these barriers to understanding, such as natural electron tunneling, coupling of electron tunneling to light capture and proton exchange as well as simpler bond breaking redox catalysis. Hope for similar solutions of more complex bioinorganic enzymes is indicated in several papers presented in this Discussion. Armed with an engineering understanding of mechanism, the current serious frustrations to successful creation of functional artificial proteins that are rooted in protein complexity can fall away. Here we discuss the genetic and biological roots of protein complexity and show how to dodge and minimize the effects of complexity. In the best-understood cases, artificial enzymes can be designed from scratch using the simplest of protein scaffolds. PMID:21322497

Dutton, P Leslie; Moser, Christopher C

2011-01-01

408

Development and Validation of an Immunoassay for Quantification of Topoisomerase I in Solid Tumor Tissues  

PubMed Central

Background Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. Methodology/Principal Findings We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. Conclusions/Significance Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors. PMID:23284638

Pfister, Thomas D.; Hollingshead, Melinda; Kinders, Robert J.; Zhang, Yiping; Evrard, Yvonne A.; Ji, Jiuping; Khin, Sonny A.; Borgel, Suzanne; Stotler, Howard; Carter, John; Divelbiss, Raymond; Kummar, Shivaani; Pommier, Yves; Parchment, Ralph E.; Tomaszewski, Joseph E.; Doroshow, James H.

2012-01-01

409

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

PubMed

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

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

2011-12-01

410

Poly(o-phenylenediamine)-carried nanogold particles as signal tags for sensitive electrochemical immunoassay of prolactin.  

PubMed

A novel class of redox-active molecular tags, poly(o-phenylenediamine)-carried nanogold particles (GPPDs), was first synthesized and functionalized with horseradish peroxidase-anti-prolactin conjugates (HRP-anti-PRL). Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of prolactin (PRL) by using GPPD-labeled HRP-anti-PRL conjugates as molecular tags on anti-PRL antibody-modified glassy carbon electrode. Compared with pure gold nanoparticles and poly(o-phenylenediamine) microspheres, the as-prepared GPPDs increased the surface coverage of the nanostructures, and enhanced the immobilization amount of biomolecules. Several labeling protocols compromising GPPD-labeled HRP-anti-PRL, nanogold particles-labeled HRP-anti-PRL and poly(o-phenylenediamine) microspheres-labeled HRP-anti-PRL, were investigated for detection of PRL, and improved analytical features were obtained with the GPPD-based strategy. With the GPPD labeling method, dependence of the electrochemical signals on the incubation time and pH of the assay solution were also studied. The strong attachment of HRP-anti-PRL to the GPPDs resulted in a good repeatability and intermediate reproducibility down to 9.8%. The dynamic concentration range spanned from 0.5 to 180 ng mL(-1) PRL with a detection limit of 0.1 ng mL(-1) at the 3S(blank) level. No significant differences at the 95% confidence level were encountered in the analysis of 10 spiked blank cattle serum samples between the developed immunoassay and enzyme-linked immunosorbent assay method for determination of PRL. PMID:22560276

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

2012-05-30

411

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

PubMed

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

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

2014-11-15

412

Measurements of Plasma Metanephrines by Immunoassay versus LC-MS/MS for Diagnosis of Pheochromocytoma.  

PubMed

Background: Reports conflict concerning measurements of plasma metanephrines for diagnosis of pheochromocytomas/paragangliomas (PPGL) by immunoassays compared to other methods. We aimed to compare the performance of a commercially available enzyme-linked immunoassay (EIA) with liquid chromatography tandem mass spectrometric (LC-MS/MS) measurements of metanephrines to diagnose PPGLs. Methods: In a sub-study of a prospective, multicenter trial to study the biochemical profiles of monoamine-producing tumors, we included 341 patients (174 males, 167 females) with suspected PPGL (median age 54 years), of whom 54 had confirmed PPGL. Plasma metanephrines were measured by EIA and LC-MS/MS, each in a specialized laboratory. Results: Plasma normetanephrine and metanephrine were measured 60% and 39% lower by EIA than by LC-MS/MS. Using upper cut-offs stipulated for the EIA, diagnostic sensitivity was only 74.1% at a specificity of 99.3%. In contrast, use of similar cut-offs for metanephrine and overall lower age-adjusted cut-offs for normetanephrine measured by LC-MS/MS returned a diagnostic sensitivity and specificity of 98.1% and 99.7%. Areas under receiver-operating characteristic curves, nevertheless, indicated comparable diagnostic performance of the EIA (0.993) and LC-MS/MS (0.985). Diagnostic sensitivity for the EIA increased to 96.2% with minimal loss in specificity (95.1%) following use of cut-offs for the EIA adapted to correct for the negative bias. Conclusions: EIA underestimates plasma metanephrines and diagnostic sensitivity is poor using commonly stipulated cut-offs, resulting in high risk for missing patients with PPGLs. Correction of this shortcoming can be achieved by appropriately determined cut-offs resulting in comparable diagnostic performance of EIA and LC-MS/MS assays. PMID:25452465

Weismann, Dirk; Peitzsch, Mirko; Raida, Anna; Prejbisz, Aleksander; Gosk, Maria; Riester, Anna; Willenberg, Holger S; Klemm, Reiner; Manz, Georg; Deutschbein, Timo; Kroiss, Matthias; Därr, Roland; Bidlingmaier, Martin; Januszewicz, Andrzej; Eisenhofer, Graeme; Fassnacht, Martin

2014-12-01

413

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

PubMed

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

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

2014-09-01

414

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

PubMed

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

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

2000-03-01

415

Multiplex Fluorescent Immunoassay for Detection of Mice Infected with Lactate Dehydrogenase Elevating Virus  

PubMed Central

Commercially available diagnostic tools for the detection of lactate dehydrogenase elevating virus (LDV) infection have been restricted to measurement of serum lactate dehydrogenase (LDH) activity levels and detection of the viral genome by RT-PCR assays. Serologic diagnosis of LDV infection has not been widely adopted due to the belief that the formation of antigen–antibody complexes and B-cell polyclonal activation may confound interpretation of results. In the current study, we inoculated BALB/c, C57BL/6, and Swiss Webster mice with LDV to compare the diagnostic reliability of a commercially available multiplex fluorescent immunoassay for the detection of antiLDV antibodies with that of the LDH enzyme assay. The serologic assay was vastly more sensitive and specific than was the LDH enzyme assay. Moreover, the serologic assay detected antiviral antibodies throughout the 3-mo time course of this study. These results suggest that antigen–antibody complex formation and polyclonal B-cell activation had little effect on assay performance. PMID:23849407

Adams, Veronica; Myles, Matthew H

2013-01-01

416

Inhibition of HIV-1 infectivity through an innate mechanism involving naturally occurring IgM anti-leukocyte autoantibodies.  

PubMed

In prior studies, we show that naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA) bind to CD3, CD4, CCR5, and CXCR4 receptors. These observations prompted us to determine whether IgM-ALA have a role in inhibiting HIV-1 infectivity by inhibiting viral entry into cells. We show that purified IgM, but not IgG, from individual sera of both normal and HIV-1 infected individuals is highly inhibitory (>95%) to HIV-1 viral infectivity both in vitro using PHA plus IL-2 activated PBL and in vivo using the human PBL-SCID mouse. Inhibition was observed with physiological doses of purified serum IgM and even after IgM was added 3 days postinfection in the in vitro assays. Absorbing purified serum IgM either with leukocytes or immobilized recombinant CD4 significantly decreased (>80%) the inhibitory effect on HIV-1 infectivity. IgM inhibited by >90% syncytia formation with the X4-IIIB infected SupT-1 cells indicating therefore that IgM inhibits viral attachment to core-receptors. IgM mediated anti-HIV-1 activity was highly specific as only certain IgM-ALA, obtained from human B cell clones inhibited HIV-1. IgM from certain HIV-1 infected individuals were not inhibitory to some R5-HIV-1 viral strains indicating that certain HIV-IgM may lack Abs reactive to strain specific coreceptor epitopes. These data indicate that an innate immune mechanism which is present from birth i.e., IgM-ALA, has a role in inhibiting HIV-1 viral entry into cells. Validation of this data with other in vivo models will be needed to determine whether in vivo administration or enhancement of IgM-ALA, e.g., through a vaccine, could prolong the asymptomatic state in HIV-1 infected individuals. PMID:18209074

Lobo, Peter I; Schlegel, Kailo H; Yuan, Wen; Townsend, Gregory C; White, Jennifer A

2008-02-01

417

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

PubMed

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

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

2014-03-15

418

Lateral flow immunoassay using magnetoresistive sensors  

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

419

Primary enzyme quantitation  

DOEpatents

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

Saunders, G.C.

1982-03-04

420

Comparison of different immunoassays and GC-MS screening of benzodiazepines in urine.  

PubMed

A total of 53 urine samples were tested by different immunoassay methods and by gas chromatography/mass spectrometry to determine repeatability of the different methods and to assess whether the immunoassays performed on samples obtained from elderly patients of the emergency section could be considered as reliable enough for identifying a benzodiazepine consumption. Repeatability was excellent for GC/MS and good for immunoassays. The specificity was not different for the three immunoassays (96%). The sensitivity varied from 36, 64 to 75% for OnLine, RIA Immunalysis and RIA DPC, respectively. An other difference between immunoassays and GC/MS was the ability of GC/MS to detect lorazepam and low concentrations of benzodiazepines whereas immunoassays did not. PMID:9919969

Augsburger, M; Rivier, L