Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

NASA Astrophysics Data System (ADS)

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

2011-10-01

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

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

PubMed

Jadhao, Samadhan J; Anderson, Larry J

2016-01-01

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

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

NASA Astrophysics Data System (ADS)

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

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

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

NASA Astrophysics Data System (ADS)

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

1982-05-01

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.

Fluorometric enzymatic assay of L-arginine

NASA Astrophysics Data System (ADS)

Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

2017-01-01

The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

Establishment of a Simple and Convenient Method for Folic Acid Enzyme Chemiluminescence Immunoassay

NASA Astrophysics Data System (ADS)

Liu, Ting; Zeng, Ling; Yu, Zhengwei; Yang, Yanfei; Qu, Yunhuan

2018-01-01

The enzyme chemiluminescence new immunoassay for folic acid (FA) was established by competition model. Add FA samples to a microtiter plate precoated with the goat anti-mouse IgG firstly, then add enzyme abled FA and FA monoclonal antibody (McAb). The values of CLIA were measured to reflect the quantity of FA. The limit of detection(LOD) of assay is 0.37ng/mL. The assay shows good correlation during 1∼30 ng/mL with correlation coefficient 0.9976. The intra- and inter-assay coefficients of variation are 4.8 % ∼ 7.3 % and 6.1 % ∼ 12.2 %, respectively. The recovery of folic acid in serum is 90.4 %∼113.2 %. Compared with determine value clinically in chemiluminescence immunoassay kit from Roche company, the correlative equation is y = 0.9689x + 0.0228, and correlation coefficient is 0.9780. Various components and kit overall show good stabilities. This method is simple and convenient, and has low LOD value. The method has overcome the shortcomings of the present references. It is easy to apply and has broad clinical application prospect. It lays an experimental foundation for the preparation of Mc Ab against folic acid and the development of domestic kit.

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

PubMed Central

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

2014-01-01

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

Fluorometric procedures for dye tracing

USGS Publications Warehouse

Wilson, James F.; Cobb, Ernest D.; Kilpatrick, F.A.

1986-01-01

This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The advantages of dye tracing are (1) low detection and measurement limits and (2) simplicity and accuracy in measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section on aerial photography is included because of its possible use to supplement ground-level fluorometry.

Fluorometric procedures for dye tracing

USGS Publications Warehouse

Wilson, James F.

1968-01-01

This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The advantages of dye tracing are (1) low detection and measurement limits and (2) simplicity and accuracy in measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section on aerial photography is included because of its possible use to supplement ground-level fluorometry.

Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water.

PubMed

Fleeker, J

1987-01-01

Two solid-phase enzyme immunoassays were developed to measure 2,4-dichlorophenoxyacetic acid (2,4-D), using 2 sets of structurally distinct immunogens and enzyme ligands. The 2,4-D analog, 2-methyl-4-chlorophenoxyacetic acid (MCPA), gave a similar response with both methods, whereas other phenoxy herbicides cross-reacted differently. In method A, the aromatic moiety of 2,4-D was distal from the carrier protein and labeled enzyme, whereas in method B, the acetic acid portion of the herbicide was distal. The use of both methods to screen for this herbicide in ground water and municipal and river water reduced the number of false-positive responses. Water sources having a low background response could be monitored with either method alone. When a concentration step, with disposable C18 extraction columns, was used, the limit of sensitivity was 5 micrograms/L. Method A was the more sensitive of the 2 methods with a limit of detection of 10 micrograms/L without the concentration step.

Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

PubMed

Groome, N P

1981-10-01

The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

Fluorometric procedures for dye tracing

USGS Publications Warehouse

Wilson, James E.; Cobb, Ernest D.; Kilpatrick, Frederick A.

1984-01-01

This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The outstanding characteristics of dye tracing are: (1) the low detection and measurement limits, and (2) the simplicity and accuracy of measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a general guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section is included on aerial photography because of its possible use to supplement ground-level fluorometry.

Evaluation of automated enzyme immunoassays for five anticonvulsants and theophylline adapted to a centrifugal analyzer.

PubMed

Urquhart, N; Godolphin, W; Campbell, D J

1979-05-01

We report a clinical evaluation of the enzyme immunoassay (EMIT) performed with the GEMSAEC centrifugal analyzer as compared to gas-liquid and liquid chromatography for anticonvulsant drugs and theophylline, respectively. A good correlation was obtained for all drugs, although some difficulties were experienced with one lot of reagent for ethosuximide. The analyzer has an economic advantage if many samples are being analyzed for few drugs in each sample.

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

PubMed

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

2006-03-01

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

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

PubMed Central

Crouch, C F

1995-01-01

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

Total Triiodothyronine by Fluorescence Polarization Immunoassay (FPIA),

DTIC Science & Technology

Graves ’ disease . Traditionally, radioimmunoassays (RIA) have been employed for the determination of total T3. Enzyme immunoassays (EIA) and fluorescence immunoassays (FIA) have been developed for many of the analytes that formerly were measured using RIA. One variation of this new generation of immunoassays is fluorescence polarization. A fluorescence polarization immunoassay (FPIA) method for total T3 has been automated by adaptation to the TDx (Abbott, Chicago, IL) clinical analyzer. The TDx total T3 assay has been evaluated as a replacement for an RIA total T3

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

PubMed Central

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

2003-01-01

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

Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.

PubMed

Milnerowicz, Halina; Bizoń, Anna

2010-01-01

Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate).

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

NASA Astrophysics Data System (ADS)

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

1994-03-01

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

Novel photoluminescence enzyme immunoassay based on supramolecular host-guest recognition using L-arginine/6-aza-2-thiothymine-stabilized gold nanocluster.

PubMed

Wang, Youmei; Lu, Minghua; Tang, Dianping

2018-06-30

A new photoluminescence (PL) enzyme immunoassay was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) via an innovative enzyme substrate, 6-aza-2-thiothymine-stabilized gold nanocluster (AAT-AuNC) with L-arginine. The enzyme substrate with strong PL intensity was formed through supramolecular host-guest assembly between guanidine group of L-arginine and AAT capped on the surface of AuNC. Upon arginase introduction, the captured L-arginine was hydrolyzed into ornithine and urea, thus resulting in the decreasing PL intensity. Based on this principle, a novel competitive-type immunoreaction was first carried out on AFB 1 -bovine serum albumin (AFB 1 -BSA) conjugate-coated microplate, using arginase-labeled anti-AFB 1 antibody as the competitor. Under the optimum conditions, the PL intensity increased with the increment of target AFB 1 , and allowed the detection of the analyte at concentrations as low as 3.2 pg mL -1 (ppt). Moreover, L-arginine-AAT-AuNC-based PL enzyme immunoassay afforded good reproducibility and acceptable specificity. In addition, the accuracy of this methodology, referring to commercial AFB 1 ELISA kit, was evaluated to analyze naturally contaminated or spiked peanut samples, giving well-matched results between two methods, thus representing a useful scheme for practical application in quantitative monitoring of mycotoxins in foodstuff. Copyright © 2018 Elsevier B.V. All rights reserved.

Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus.

PubMed

Katz, J B; Hanson, S K

1987-02-01

A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS). The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen. Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary. A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT). CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85). Relative to the SNT, CELIA sensitivity was 100%; specificity was 76%. CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT. Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed. A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made. It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

PubMed

Kai, Junhai; Puntambekar, Aniruddha; Santiago, Nelson; Lee, Se Hwan; Sehy, David W; Moore, Victor; Han, Jungyoup; Ahn, Chong H

2012-11-07

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

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

PubMed

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

2015-01-01

Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. © 2015 by The American Society of Hematology.

Determination of chondroitin-6-sulphate by a competitive enzyme immunoassay using a biotinylated antigen.

PubMed

Kähnert, H; Brinkmann, T; Gässler, N; Kleesiek, K

1994-04-01

A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitin-6-sulphate in the samples and the biotinylated antigen. This assay enables the quantification of chondroitin-6-sulphate in the low concentration range of 16-120 micrograms/l. The intra-assay and inter-assay coefficients of variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/l phosphate-buffered saline, an albumin solution (40 g/l in phosphate-buffered saline) and cell culture medium (containing 100 ml/l foetal calf serum). Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal range was 55-169 micrograms/l. In men the mean value was estimated at 102.2 +/- 37.1 micrograms/l and in women at 98.7 +/- 26.4 micrograms/l. No age or sex dependence was observed. The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 +/- 21.1 mg/kg creatinine (mean +/- standard deviation) and in females (n = 10) 53.5 +/- 21.3 mg/kg creatinine. The clearance rate in men was 0.41 +/- 0.22 ml x min-1 and in women 0.38 +/- 0.15 ml x min-1. No sex dependence was found. Furthermore, the enzyme immunoassay was modified to measure the specific incorporation of a radioactively labelled precursor ([14C]galactosamine) into chondroitin-6-sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)

IMMUNOASSAY HUMAN EXPOSURE STUDIES

EPA Science Inventory

The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

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

NASA Technical Reports Server (NTRS)

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

1987-01-01

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

Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

PubMed

Tabachnick, W J; Mecham, J O

1991-03-01

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

Combining supercritical fluid extraction of soil herbicides with enzyme immunoassay analysis.

PubMed

Stearman, G K

2001-10-01

Supercritical fluid extraction (SFE) of soil herbicides followed by enzyme immunoassay analysis (EIA) is explained in a step-by-step process. Extracted herbicides, include 2,4-D, simazine, atrazine, and alachlor. The herbicide, trifluralin was not successfully analyzed by EIA because of crossreacting metabolites. Problems with SFE, including uneven packing of cells, leaks, uneven flow and clogging, can largely be eliminated as the method parameters are optimized. It was necessary to add modifiers including methanol or acetone to the SF CO2 to increase the solubility of the analytes. Detection limits of 2.5 ng/g soil for atrazine and alachlor and 15 ng/g soil for simazine and 2,4-D without concentration of the sample were achieved. Recoveries above 80% and relative standard deviations (RSDs) less than 15% for 2,4-D simazine, atrazine and alachlor were achieved. Atrazine and alachlor recoveries were above 90% with RSDs below 10%. Forty soil samples could be extracted and analyzed in an 8-h day.

Immunoassays for pesticide monitoring

NASA Astrophysics Data System (ADS)

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

1995-05-01

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

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

PubMed

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

2015-05-01

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

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2.

PubMed

Sakamoto, Misato; Shoji, Atsushi; Sugawara, Masao

2016-07-15

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

Ovine echinococcosis I. Immunological diagnosis by enzyme immunoassay.

PubMed

Gatti, Antonio; Alvarez, Angela Rosa; Araya, Daniel; Mancini, Sergio; Herrero, Eduardo; Santillan, Graciela; Larrieu, Edmundo

2007-01-31

Immunodiagnosis in sheep presents problems of sensitivity and specificity, limiting its applicability in surveillance systems. The objective of this study was to develop a sensitive, specific and accessible technique for diagnosing cystic echinococcosis in naturally infected sheep and to evaluate the validity of necropsy as a reference test. A total of 247 sheep were studied at slaughterhouses, confirming the parasitological diagnosis with histology. Serum was processed with enzyme immunoassay (EIA) using three antigen preparations: total hydatid liquid (LHT), purified fraction of LHT (S2B) and purified lipoprotein (B). Western Blot (WB) was used as a control. EIA proved effective for differentiating Echinococcus granulosus from larval stage of Taenia hydatigena and intestinal cestodes in all three antigen preparations. Serums from macroscopically negative sheep were reactive to EIA and positive with WB. In the whole flock, sensitivity was 89.2% for LHT, 80.0% for S2B and 86.4% for B. Sensitivity in lambs was 78.6% for LHT, 75.0% for S2B and 64.3% for B. Macroscopic diagnosis at the time of slaughter was found to have limitations as a reference test for immunodiagnosis of cystic equinococcosis in sheep, so it was necessary to include histology and WB as reference tests. LHT was the antigen preparation of greatest value and EIA proved to be a sensitive and specific technique, adequate for surveillance systems and for evaluating control programmes.

Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

PubMed Central

Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

2011-01-01

A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

Automated homogeneous liposome immunoassay systems for anticonvulsant drugs.

PubMed

Kubotsu, K; Goto, S; Fujita, M; Tuchiya, H; Kida, M; Takano, S; Matsuura, S; Sakurabayashi, I

1992-06-01

We developed automated homogeneous immunoassays, based on immunolysis of liposomes, for measuring phenytoin, phenobarbital, and carbamazepine from serum. Liposome lysis was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. The procedure was fully automated on a routine automated clinical analyzer. Within-run, between-run, dilution, and recovery tests showed good accuracies and reproducibilities. Bilirubin, hemoglobin, triglycerides, and Intrafat did not affect assay results. The results obtained by liposome immunoassays for phenytoin, phenobarbital, and carbamazepine correlated well with those obtained by enzyme-multiplied immunoassay (Syva EMIT) kits (r = 0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsant drug concentrations in blood.

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

PubMed Central

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

2012-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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...

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

PubMed

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

2014-06-01

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.

Application of microstructural optical waveguides with hollow core for enzyme immunoassay

NASA Astrophysics Data System (ADS)

Pidenko, Pavel S.; Pidenko, Sergei A.; Burmistrova, Natalia A.; Shuvalov, Andrei A.; Chibrova, Anastasiya A.; Skibina, Yulia S.; Goryacheva, Irina Y.

2018-04-01

Microstructural optical waveguides with the hollow core are actively studied as a promising support for heterogeneous immunoassay in development of new optical biosensor elements for medicine and biology. Overcoming of the limitations associated with the low sorption capacity of glass used for the waveguides production is a crucial step for this assay format. In this work the possibility of silanization of microstructural optical waveguides with the hollow core using (3-glycidyloxypropyl) trimethoxysilane and their further application to enzymatic immunoassay was studied.

Kinase Activity Studied in Living Cells Using an Immunoassay

ERIC Educational Resources Information Center

Bavec, Aljos?a

2014-01-01

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

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

PubMed Central

Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

2015-01-01

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

Ultramicro-fluorometric assay for the diagnosis of Gaucher disease in dried blood spots on filter paper.

PubMed

Herrera, D; Monaga, M; Campos, D; Pampín, Y; González, E C; Lavaut, K

2013-01-01

Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency of the lysosomal acid β-D-glucosidase (GBA). The aim of this study was to develop an ultramicro-fluorometric assay based on the method of Chamoles et al. for determining GBA activity in dried blood spots on filter paper (DBS). The assay used 3-mm diameter blood spot and 8 mmol/l of 4-methylumbelliferyl-β-D-glucoside as enzymatic substrate. The reaction occurred in plates incubated at 37°C for 20 hours and the enzyme activity was expressed in μmol hydrolysed substrate/l blood/h. The fluorescence of the enzyme product was automatically measured in a fluorometer-photometer reader (SUMA Technology). The intra and inter-assay coefficients of variation were lower than 9 and 12%, respectively, and the recovery range was 97-109%.Three patients with GD were correctly diagnosed using the ultramicroassay. Healthy newborn DBS samples (n = 3003) from the National Neonatal Screening Program were analyzed, and the mean GBA activity was 5.7 μmol/l blood/h. Our assay showed high Pearson (n = 26; r = 0.99) and concordance correlations (ρc = 0.99) with the traditional method described by Chamoles et al. The analytical performance characteristics of our ultramicro-fluorometric assay suggest that it can be used in the diagnosis of GD in newborns and adults.

Erythema migrans and serodiagnosis by enzyme immunoassay and immunoblot with three borrelia species.

PubMed

Stanek, G; Breier, F; Menzinger, G; Schaar, B; Hafner, M; Partsch, H

1999-12-10

There is wide divergence of opinion between physicians regarding the use of serological measures for the diagnosis and treatment of erythema migrans, the hallmark of Lyme borreliosis. We studied the outcome of an enzyme immunoassay and immunoblot (Western blot) used on the sera of patients who had suffered tick bite and erythema migrans, and had been subsequently treated with various antibiotics. Ninety-nine consecutive patients presenting with erythema migrans after tick bite were prospectively recruited at the outpatient department of two Vienna City hospitals and at the consultation office for Lyme borreliosis of the Institute of Hygiene. University Vienna. Blood samples were taken before antibiotic treatment and 3 and 6 months thereafter. Blood samples from 100 blood donors served as controls. Antibodies against Borrelia burgdorferi sensu lato were determined by enzyme immunoassay (IgG and IgM EIA) and by IgG immunoblot. The latter was performed with isolates of B. alzelii (H2) B. burgdorferi sensu stricto (Le) and B. garinii (W) from Austrian patients. The 4 interpretation criteria for immunoblot results were: A (3 bands out of 8), B (2 bands out of 9), C and D (1 band out of 6). In all patients, the erythema resolved within the treatment period. No complications secondary to the borrelia infection were registered. After treatment there was no significant change in titre, nor was there a difference in the immunoblot pattern between the first, second and third serum samples. Serum antibodies to B. burgdorferi were positive by EIA in 22.9% (IgG) and 2.5% (IgM). Immunoblot results offered by borrelia species and by the interpretation criteria, ranging between 8.3% (criterion A, strain Le) and 44.2% (criterion D, strain H2). By EIA, control samples were IgG and IgM positive in 5% and 1%, respectively. Positive immunoblot results with strain H2 were found in 9%, 13%, 18%, and 20% by the criteria A through D respectively. After antibiotic treatment of erythema

Enzyme immunoassay for tenuazonic acid in apple and tomato products.

PubMed

Gross, Madeleine; Curtui, Valeriu; Ackermann, Yvonne; Latif, Hadri; Usleber, Ewald

2011-12-14

The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

PubMed

Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

2015-01-01

The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

PubMed

Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

1993-08-01

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Impact of HIV Subtype on Performance of the Limiting Antigen-Avidity Enzyme Immunoassay, the Bio-Rad Avidity Assay, and the BED Capture Immunoassay in Rakai, Uganda

PubMed Central

Longosz, Andrew F.; Serwadda, David; Nalugoda, Fred; Kigozi, Godfrey; Franco, Veronica; Gray, Ronald H.; Quinn, Thomas C.; Eshleman, Susan H.

2014-01-01

Abstract Previous studies demonstrated that individuals with subtype D HIV infection who had been infected for 2 or more years were frequently misclassified as assay positive using cross-sectional incidence assays. Samples from 510 subjects (212 subtype A, 298 subtype D) who were infected for 2.2 to 14.5 years (median 5.4 years) and were not virally suppressed were tested using an LAg-Avidity enzyme immunoassay (LAg-Avidity EIA), Bio-Rad Avidity assay, and BED capture enzyme immunoassay (BED-CEIA). The performance of these three assays was evaluated using various assay cutoff values [LAg-Avidity EIA: <1.0 OD-n and <2.0 OD-n; Bio-Rad Avidity assay: <40% avidity index (AI) and <80% AI; BED-CEIA: <0.8 OD-n]. The mean LAg-Avidity EIA result was higher for subtype A than D (4.54±0.95 vs. 3.86±1.26, p<0.001); the mean Bio-Rad Avidity assay result was higher for subtype A than D (88.9%±12.5% vs. 75.1±30.5, p<0.001); and the mean BED-CEIA result was similar for the two subtypes (2.2±1.2 OD-n for subtype A, 2.2±1.3 OD-n for subtype D, p<0.9). The frequency of misclassification was higher for individuals with subtype D infection compared to those with subtype A infection, using either the LAg-Avidity EIA with a cutoff of <2.0 OD-n or the Bio-Rad Avidity assay with cutoffs of <40% or <80% AI. No subtype-specific differences in assay performance were observed using the BED-CEIA. Sex and age were not significantly associated with misclassification by any assay. The LAg-Avidity EIA with a cutoff <1.0 OD-n had the lowest frequency of misclassification in this Ugandan population. PMID:24083837

Fluorometric prediction of successful amputation level in the ischemic limb.

PubMed

Silverman, D G; Rubin, S M; Reilly, C A; Brousseau, D A; Norton, K J; Wolf, G L

1985-01-01

The present study was undertaken to compare fluorometric documentation of fluorescein dye delivery with the standard means of determining the level at which an amputation should be performed in the dysvascular extremity. Thirty-nine patients underwent lower-extremity amputation at the level determined by the surgeon based upon physical examination, angiography, segmental pressure indices, and/or pulse volume recordings. In addition, fiberoptic fluorometry was performed preoperatively. After intravenous administration of sodium fluorescein (4-8 mg/kg), fluorometric readings were obtained by placing the fluorometer's light guide on 126 reading sites. Fluorometric findings were evaluated retrospectively, and therefore did not influence the surgeon's decision. Of the 39 amputations performed overall, only 26 healed. The accuracy of the standard criteria was lowest for the 20 below-ankle amputations, where only 12 cases healed. Alternatively, fluorometric indices separated healing from nonhealing sites in 36 of the 39 cases and in 18 of the 20 below-ankle amputations. Overall, healing sites averaged 94 percent of the fluorescence of the healthy reference area, while nonhealing sites averaged only 29 percent. We conclude that fluorometry should prove to be a valuable adjunct in the assessment of the dysvascular extremity. It uses a low dose of dye, is easy to perform, and is readily repeatable.

Fluorometric Biosniffer Camera "Sniff-Cam" for Direct Imaging of Gaseous Ethanol in Breath and Transdermal Vapor.

PubMed

Arakawa, Takahiro; Sato, Toshiyuki; Iitani, Kenta; Toma, Koji; Mitsubayashi, Kohji

2017-04-18

Various volatile organic compounds can be found in human transpiration, breath and body odor. In this paper, a novel two-dimensional fluorometric imaging system, known as a "sniffer-cam" for ethanol vapor released from human breath and palm skin was constructed and validated. This imaging system measures ethanol vapor concentrations as intensities of fluorescence through an enzymatic reaction induced by alcohol dehydrogenase (ADH). The imaging system consisted of multiple ultraviolet light emitting diode (UV-LED) excitation sheet, an ADH enzyme immobilized mesh substrate and a high-sensitive CCD camera. This imaging system uses ADH for recognition of ethanol vapor. It measures ethanol vapor by measuring fluorescence of nicotinamide adenine dinucleotide (NADH), which is produced by an enzymatic reaction on the mesh. This NADH fluorometric imaging system achieved the two-dimensional real-time imaging of ethanol vapor distribution (0.5-200 ppm). The system showed rapid and accurate responses and a visible measurement, which could lead to an analysis of metabolism function at real time in the near future.

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

PubMed

Bi, Sai; Zhou, Hong; Zhang, Shusheng

2009-06-15

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.

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

PubMed

Yang, Xinjian; Gao, Zhiqiang

2015-04-25

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

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

USGS Publications Warehouse

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

2001-01-01

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

SITE EVALUATION OF FIELD PORTABLE PENTACHLOROPHENOL IMMUNOASSAYS

EPA Science Inventory

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

Rapid quantitative analysis of 8-iso-prostaglandin-F(2alpha) using liquid chromatography-tandem mass spectrometry and comparison with an enzyme immunoassay method.

PubMed

Dahl, Jeffrey H; van Breemen, Richard B

2010-09-15

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the measurement of urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a biomarker of lipid peroxidation. Because urine contains numerous F(2) prostaglandin isomers, each with identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF(2alpha) from its isomers is necessary for its quantitative analysis using MS/MS. We were able to achieve this separation using an isocratic LC method with a run time of less than 9min, which is at least threefold faster than previous methods, while maintaining sensitivity, accuracy, precision, and reliability. The limits of detection and quantitation were 53 and 178pg/ml urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays to be in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS/MS. Our method was optimized for rapid measurement of 8-iso-PGF(2alpha) in urine, and it is ideally suited for clinical sample analysis. 2010 Elsevier Inc. All rights reserved.

Capillary electrophoresis-based immunoassays: principles and quantitative applications.

PubMed

Moser, Annette C; Hage, David S

2008-08-01

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

Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

PubMed Central

Harmsen, T; Jongerius, M C; van der Zwan, C W; Plantinga, A D; Kraaijeveld, C A; Berbers, G A

1992-01-01

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA. PMID:1500523

The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

PubMed

Siennicka, Joanna; Częścik, Agnieszka; Trzcińska, Agnieszka

2014-01-01

The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

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

PubMed

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

2014-05-20

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 (λ = 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

Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.

PubMed Central

Garcia, L S; Shimizu, R Y

1997-01-01

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

PubMed

Wegner, S; Bauer, J I; Dietrich, R; Märtlbauer, E; Usleber, E; Gottschalk, C; Gross, M

2017-02-01

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml -1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins. © 2016 The Society for Applied Microbiology.

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

PubMed Central

Bouzas, Lorena; Hermida, Jesús

2009-01-01

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

Inclusion of fluorophores in cyclodextrins: a closer look at the fluorometric determination of association constants by linear and nonlinear fitting procedures

NASA Astrophysics Data System (ADS)

Hutterer, Rudi

2018-01-01

The author discusses methods for the fluorometric determination of affinity constants by linear and nonlinear fitting methods. This is outlined in particular for the interaction between cyclodextrins and several anesthetic drugs including benzocaine. Special emphasis is given to the limitations of certain fits, and the impact of such studies on enzyme-substrate interactions are demonstrated. Both the experimental part and methods of analysis are well suited for students in an advanced lab.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

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

2006-11-01

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

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

PubMed

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

2014-07-01

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of lithium in rocks: Fluorometric method

USGS Publications Warehouse

White, C.E.; Fletcher, M.H.; Parks, J.

1951-01-01

The gravimetric method in general use for the determination of lithium is tedious, and the final weighed product often contains other alkali metals. A fluorometric method was developed to shorten the time required for the analysis and to assure that the final determination is for lithium alone. This procedure is based on the complex formed between lithium and 8-hydroxyquinoline. The fluorescence is developed in a slightly alkaline solution of 95% alcohol and measurement is made on a photoelectric fluorometer. Separation from the ore is carried out by the wet method or by the distillation procedure. Sodium and potassium are removed by alcohol and ether, but complete separation is not necessary. Comparison of analyzed samples shows excellent agreement with spectrographic and gravimetric methods. The fluorometric method is more rapid than the gravimetric and produces more conclusive results. Another useful application is in the preparation of standard lithium solutions from reagent quality salts when a known standard is available. In this case no separations are necessary.

Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2007-07-01

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

Performance of an organic photodiode as an optical detector and its application to fluorometric flow-immunoassay for IgA.

PubMed

Miyake, Mayo; Nakajima, Hizuru; Hemmi, Akihide; Yahiro, Masayuki; Adachi, Chihaya; Soh, Nobuaki; Ishimatsu, Ryoichi; Nakano, Koji; Uchiyama, Katsumi; Imato, Toshihiko

2012-07-15

The performance of an organic thin film photodiode (OPD), fabricated from a hetero-junction comprised of two layers of C(60) and a phthalocyanine-Cu(II) complex was evaluated by detecting the chemiluminescence generated from the reaction of luminol with horseradish peroxidase in the presence of H(2)O(2), and the fluorescence from resorufin, as an optical detector. The photocurrent of the OPD was linear with respect to the power of light from a commercial LED. The sensitivity of the OPD was sufficient for detecting chemiluminescence with a power 0.1μW/cm(2). The OPD was successfully used in a flow-immunoassay for IgA, a marker of human stress, in which a sandwich immunoassay was carried out on the microchip and the fluorescence from resorufin, produced by the enzymatic reaction, was detected. The detection limits for resorufin and IgA were 5.0μM and 16ng/mL, respectively. The photosensitivity of the OPD remained relatively constant for a minimum of one year. Copyright © 2012 Elsevier B.V. All rights reserved.

Measurement of ring A-reduced progesterone metabolites by enzyme-linked immunoassay with colorimetric detection: baseline levels of six metabolites, including pregnanolone, in male rat plasma.

PubMed

Ocvirk, Rok; Franklin, Keith B J; Pearson Murphy, Beverley E

2009-02-01

The performance of an antiserum to progesterone and pregnane neurosteroids was assessed in two competitive assay setups: radioimmunoassay and enzyme-linked immunoassay with colorimetric detection, both with the same limit of detection of 2 pg. The enzyme-linked immunoassay was less labor-intensive and had better precision of measurement and was used to measure progesterone and six of its ring A-reduced metabolites in rat plasma. The measured levels of allopregnanolone and progesterone were in agreement with those reported previously when measured by gas chromatography/mass spectrometry and high-performance liquid chromatography coupled with radioimmunoassay and substantially lower than those previously measured by radioimmunoassay without chromatographic separation. Both isomers of dihydroprogesterone and all four isomers of pregnanolone were detected in rat plasma, indicating that progesterone is metabolized by reduction at the C5 and C3 position of the A ring, in both alpha and beta configurations. In addition to 5beta-dihydroprogesterone and isopregnanolone, which have not been previously detected in the rat, we found considerable amounts of pregnanolone, which is neuroactive, with similar potency to that of allopregnanolone but was previously thought not to be produced in rats.

Immunoassays

NASA Astrophysics Data System (ADS)

Hsieh, Y.-H. Peggy

Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

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

PubMed

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

2013-08-01

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

Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria.

PubMed

Engler, K H; Efstratiou, A

2000-04-01

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.

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

PubMed Central

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

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

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

PubMed

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

2014-08-15

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. Copyright © 2014 Elsevier Inc. All rights reserved.

Substrates and method for determining enzymes

DOEpatents

Smith, Robert E.; Bissell, Eugene R.

1981-01-01

A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.

Substrates and method for determining enzymes

DOEpatents

Smith, R.E.; Bissell, E.R.

1981-10-13

A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, J.W.; Grindon, A.J.; Feorino, P.M.

1986-07-18

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

Immunoassay as an analytical tool in agricultural biotechnology.

PubMed

Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

2006-01-01

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice.

PubMed

Yang, Xi; Zhou, Tao; Yu, Lei; Tan, Wenwen; Zhou, Rui; Hu, Yonggang

2015-03-01

A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd.

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

PubMed

Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

2012-04-11

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.

Optimizing the fluorometric β-glucuronidase assay in ruminant milk for a more precise determination of mastitis.

PubMed

Larsen, Torben; Aulrich, Karen

2012-02-01

Activity of the enzyme β-glucuronidase (EC 3.2.1.31) is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination may therefore be less precise and the analytical procedure very time consuming and labour intensive. The present study optimized the fluorometric determination of the β-glucuronidase activity with respect to substrate concentration, pH, incubation time etc., validated the assay, and developed it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. β-glucuronidase, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and N-acetyl-β-d-glucosaminidase (NAGase) activity, and for somatic cell counts (SCC) and the variables were compared. Activity of β-glucuronidase was moderately but significantly correlated to SCC (r=0·21; n=768) as well as the other mentioned variables (r=0·25-0·43; n=825). Simple indices based on β-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether β-glucuronidase can compete with well-established indicators of mastitis in cows such as LDH or NAGase as well as determine whether β-glucuronidase activity, in combination with other indicators of mastitis, has an advantage. Nineteen milk samples from subclinical and latent cases of mastitis (individual quarters) were identified for specific pathogens (PCR method) and measured for β-glucuronidase activity. The activity was tested at four different pH levels (5·5, 6·0, 6·5 and 7·0) in order to investigate the possibility of

Detection of anti-nuclear antibody by immunofluorescence assay and enzyme immunoassay in childhood systemic lupus erythematosus: experience from Bangladesh.

PubMed

Dipti, Tanjeem Rabika; Azam, Mohammad Shaiful; Sattar, Mohammad Humayun; Rahman, Shahana Akhter

2012-02-01

Systemic lupus erythematosus (SLE) is a multisystem, chronic but often episodic, autoimmune disease that is characterized by the presence of antinuclear antibodies (ANA). The criteria set by American College of Rheumatology are widely used for diagnosis of SLE. Elevation of ANA titer is the most sensitive of the ACR criteria. There are different methods for detection of ANA. Indirect immunofluorescence (ANA-IFA) and enzyme immunoassay (ANA-EIA) are commonly used methods. The sensitivity of ANA-IFA using HEp-2 cell substrate is 90-100% in systemic rheumatic diseases. In Bangladesh most of the laboratories use ANA-EIA for detection of ANA. As the sensitivity of ANA-EIA is lower than ANA-IFA it might be that we are missing many cases of ANA positivity in childhood SLE cases. To detect ANA by immunofluorescence assay using HEp-2 cell substrate and enzyme immunoassay in childhood SLE and to compare the diagnostic performance of these methods. This is a cross-sectional analytical study. A total of 40 patients were enrolled. Among them 20 were childhood SLE cases. Another 20 patients of childhood rheumatic diseases other than SLE were taken as the disease control group. In childhood SLE cases, 100% were ANA-positive by IFA and 55% were ANA positive by EIA. The sensitivity of ANA-IFA was 100%. In contrast, sensitivity of ANA-EIA was 55%.   ANA-IFA is superior to ANA-EIA for detection of ANA in childhood SLE patients. ANA-IFA should be the primary screening test for children with clinical features suggestive of SLE. © 2011 The Authors. International Journal of Rheumatic Diseases © 2011 Asia Pacific League of Associations for Rheumatology and Blackwell Publishing Asia Pty Ltd.

Novel silver nanoparticle-enhanced fluorometric determination of trace tetracyclines in aqueous solutions.

PubMed

Wang, Ping; Wu, Tun-Hua; Zhang, Yong

2016-01-01

Metal-enhanced fluorescence (MEF) has exhibited promise for applications in fluorometric assays. The effects of silver nanoparticles (AgNP) on the fluorescence behaviours of tetracycline hydrochloride (TCH) and chlortetracycline hydrochloride (CTC) in aqueous solutions were investigated. The experimental results demonstrated that the fluorescence intensities of each tetracycline in water solutions were greatly enhanced by AgNP through the MEF effect. In addition, a novel silver nanoparticle-enhanced fluorometric method was established for the direct determination of TCH and CTC in aqueous solutions. Under optimum experimental conditions, the linear dynamic ranges for the determination of TCH and CTC in aqueous solutions varied from 0.10 to 6.0 mg L(-1) and 0.050 to 3.0 mg L(-1) with detection limits of 0.63 µg L(-1) and 0.19 µg L(-1), respectively, and with the relative standard deviation of less than 1.9% (n=9). The experimental recovery results for the determination of TCH and CTC in aqueous solutions ranged from 93-106% and 95-104%, respectively. Compared with the established method without the addition of AgNP, the limits of quantitation of the silver nanoparticle-enhanced fluorometric method were approximately 5-fold lower for TCH and 3-fold lower for CTC. Moreover, the newly established silver nanoparticle-enhanced fluorometric method was successfully applied to the direct determination of TCH and CTC in pharmaceutical preparations. Copyright © 2015 Elsevier B.V. All rights reserved.

Colloidal nanomaterial-based immunoassay.

PubMed

Teste, Bruno; Descroix, Stephanie

2012-06-01

Nanomaterials have been widely developed for their use in nanomedicine, especially for immunoassay-based diagnosis. In this review we focus on the use of nanomaterials as a nanoplatform for colloidal immunoassays. While conventional heterogeneous immunoassays suffer from mass transfer limitations and consequently long assay time, colloidal immunosupports allow target capture in the entire volume, thus speeding up reaction kinetics and shortening assay time. Owing to their wide range of chemical and physical properties, nanomaterials are an interesting candidate for immunoassay development. The most popular colloidal nanomaterials for colloidal immunoassays will be discussed, as well as their influence on immune reactions. Recent advances in nanomaterial applications for different formats of immunoassays will be reported, such as nanomaterial-based indirect immunoassays, optical-based agglutination immunoassays, resonance energy transfer-based immunoassays and magnetic relaxation-based immunoassays. Finally, the future of using nanomaterials for homogeneous immunoassays dedicated to clinical diagnosis will be discussed.

Fluorometric imaging methods for palladium and platinum and the use of palladium for imaging biomolecules.

PubMed

Tracey, Matthew P; Pham, Dianne; Koide, Kazunori

2015-07-21

Neither palladium nor platinum is an endogenous biological metal. Imaging palladium in biological samples, however, is becoming increasingly important because bioorthogonal organometallic chemistry involves palladium catalysis. In addition to being an imaging target, palladium has been used to fluorometrically image biomolecules. In these cases, palladium species are used as imaging-enabling reagents. This review article discusses these fluorometric methods. Platinum-based drugs are widely used as anticancer drugs, yet their mechanism of action remains largely unknown. We discuss fluorometric methods for imaging or quantifying platinum in cells or biofluids. These methods include the use of chemosensors to directly detect platinum, fluorescently tagging platinum-based drugs, and utilizing post-labeling to elucidate distribution and mode of action.

Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis

PubMed Central

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

1999-01-01

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

Immunoassay

USDA-ARS?s Scientific Manuscript database

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

[Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

PubMed

Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun

2016-11-25

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

A polymerase chain reaction enzyme immunoassay for diagnosing infection caused by Aspergillus fumigatus.

PubMed Central

Golbang, N; Burnie, J P; Matthews, R C

1999-01-01

AIM: To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. METHODS: The PCR reaction was based on primers from the 18s rRNA gene. Binding of the product to a streptavidin coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR and this was the tag to which antibody was bound in the subsequent EIA. RESULTS: The optical density (OD) endpoint was < 0.1 in 10 sera from neutropenic patients with no evidence of invasive aspergillosis, and in 10 sera from nonneutropenic patients with bacterial pneumonia (group 1). The OD from five of 12 patients with allergic bronchopulmonary aspergillosis (ABPA) (group 2), three with an aspergilloma (group 3), and five with possible invasive aspergillosis (group 4) was > or = 0.1. In 63 sera from 33 cases of proven invasive aspergillosis (group 5) an OD > or = 0.1 was achieved in 48 sera from 30 patients. The maximum OD was 0.510. The level fell in survivors and gradually rose in fatal cases. CONCLUSIONS: This assay validated the concept of diagnosing invasive aspergillosis by measuring levels of circulating fungal DNA in serum. PMID:10562808

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever.

PubMed

Boisen, Matthew L; Hartnett, Jessica N; Shaffer, Jeffrey G; Goba, Augustine; Momoh, Mambu; Sandi, John Demby; Fullah, Mohamed; Nelson, Diana K S; Bush, Duane J; Rowland, Megan M; Heinrich, Megan L; Koval, Anatoliy P; Cross, Robert W; Barnes, Kayla G; Lachenauer, Anna E; Lin, Aaron E; Nekoui, Mahan; Kotliar, Dylan; Winnicki, Sarah M; Siddle, Katherine J; Gbakie, Michael; Fonnie, Mbalu; Koroma, Veronica J; Kanneh, Lansana; Kulakosky, Peter C; Hastie, Kathryn M; Wilson, Russell B; Andersen, Kristian G; Folarin, Onikepe O; Happi, Christian T; Sabeti, Pardis C; Geisbert, Thomas W; Saphire, Erica Ollmann; Khan, S Humarr; Grant, Donald S; Schieffelin, John S; Branco, Luis M; Garry, Robert F

2018-04-12

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient's clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

The effect of preparation, storage and shipping of dried blood spots on the activity of five lysosomal enzymes.

PubMed

Elbin, Carole S; Olivova, Petra; Marashio, Carla A; Cooper, Samantha K; Cullen, Emmaline; Keutzer, Joan M; Zhang, X Kate

2011-06-11

Fluorometric and tandem mass spectrometry assays can be used to measure lysosomal enzyme activities in dried blood spots (DBS). The effect of DBS preparation, storage and shipping was evaluated on the activities of acid α-glucosidase, acid α-galactosidase, acid β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase. Whole blood from normal donors was used to prepare DBS following Clinical and Laboratory Standards Institute guidelines and by several deviations. Some DBS were subjected to various treatments, storage and shipping conditions. The activity of 5 lysosomal enzymes (GAA, GLA, GBA, ASM, and GALC) was measured using tandem mass spectrometric and fluorometric (GAA only) assays with 2 distinct and commonly used synthetic substrates. Enzyme activities were strongly affected by the way DBS were prepared and stored. Exposure of DBS to elevated heat and humidity can destroy enzyme functions rapidly. DBS prepared from poorly mixed blood caused significant variation on enzyme activities. EDTA, but not heparin, as an anti-coagulant gave more precise results. The study confirmed the importance of proper and consistent DBS preparation and storage when screening for deficiencies of lysosomal enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

PubMed

Ordronneau, P; Abdullah, L H; Petrusz, P

1991-09-13

In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

PubMed

Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

2016-01-15

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

Silver nanoparticles deposited on graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.

PubMed

Yang, Lin; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng Zhi

2018-06-14

Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm-1 and 1350 cm-1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL-1 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.

Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

PubMed

Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

2017-04-01

This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm 2 of one banknote by swabbing 1cm 2 : 0, 14, and 21pg/cm 2 . Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

PubMed

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

PubMed

Chen, Chaochao; Luo, Jiaxun; Li, Chenglong; Ma, Mingfang; Yu, Wenbo; Shen, Jianzhong; Wang, Zhanhui

2018-03-21

The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

PubMed

Li, Xiangmei; Wang, Wenjun; Wang, Limiao; Wang, Qi; Pei, Xingyao; Jiang, Haiyang

2015-10-01

Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

PubMed

Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

2016-06-05

Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). Copyright © 2016 Elsevier B.V. All rights reserved.

Homogeneous Immunoassays: Historical Perspective and Future Promise

NASA Astrophysics Data System (ADS)

Ullman, Edwin F.

1999-06-01

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

[The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

PubMed

Starkova, T V; Poletaeva, O G; Kovrova, E A; Krasovskaia, N N; Tkachenko, T N; Masiago, A V; Ofitserov, V I; Tereshchenko, A Iu

2011-01-01

The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

PubMed

Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

2016-11-10

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags 1 . Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine.

PubMed

Ma, Mingfang; Wen, Kai; Beier, Ross C; Eremin, Sergei A; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

2016-07-20

We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL).

PubMed

Chen, P; Tian, Z; Digenis, G A; Tai, H H

1996-06-01

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.

A sensitive chemiluminescence enzyme immunoassay based on molecularly imprinted polymers solid-phase extraction of parathion.

PubMed

Chen, Ge; Jin, Maojun; Du, Pengfei; Zhang, Chan; Cui, Xueyan; Zhang, Yudan; She, Yongxin; Shao, Hua; Jin, Fen; Wang, Shanshan; Zheng, Lufei; Wang, Jing

2017-08-01

The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C 18 . However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay.

PubMed

Kuppers, R C; Outschoorn, I M; Hamilton, R G; Burek, C L; Rose, N R

1993-04-01

A quantitative enzyme-linked immunoassay that measures in absolute terms the subclass concentration of human thyroglobulin (huTg)-specific IgG autoantibody was developed. Unique to this study was the use of an affinity-purified anti-huTg standard with a known concentration of the four IgG subclasses. The sensitivity of the ELISA assay was 1-5 ng/ml depending on the IgG subclass being measured. We examined 22 sera of patients with autoimmune thyroid disease. The total huTg-specific antibody concentrations in serum ranged from 0 to nearly 3000 micrograms/ml of IgG. The IgG subclass distribution in individuals with low huTg-specific IgG (< 10 micrograms/ml) was primarily IgG1 and IgG3 Ab. Patients with intermediate levels of huTg IgG (10-600 micrograms/ml) expressed all four subclasses; however, no particular subclass was dominant. Individuals with > 1000 micrograms/ml also showed huTg-Ab in all four subclasses, however, IgG1 and IgG2 were dominant. All four IgG subclasses were used in the response to huTg, although the pattern of usage varied between individuals. There was no dominant subclass usage seen in this patient population.

Sandwich-type enzyme immunoassay for big endothelin-I in plasma: concentrations in healthy human subjects unaffected by sex or posture.

PubMed

Aubin, P; Le Brun, G; Moldovan, F; Villette, J M; Créminon, C; Dumas, J; Homyrda, L; Soliman, H; Azizi, M; Fiet, J

1997-01-01

A sandwich-type enzyme immunoassay has been developed for measuring human big endothelin-1 (big ET-1) in human plasma and supernatant fluids from human cell cultures. Big ET-1 is the precursor of endothelin 1 (ET-1), the most potent vasoconstrictor known. A rabbit antibody raised against the big ET-1 COOH-terminus fragment was used as an immobilized antibody (anti-P16). The Fab' fragment of a monoclonal antibody (1B3) raised against the ET-1 loop fragment was used as the enzyme-labeled antibody, after being coupled to acetylcholinesterase. The lowest detectable value in the assay was 1.2 pg/mL (0.12 pg/well). The assay was highly specific for big ET-1, demonstrating no cross-reactivity with ET-1, <0.4% cross-reactivity with big endothelin-2 (big ET-2), and <0.1% with big endothelin-3 (big ET-3). We used this assay to evaluate the effect of two different postural positions (supine and standing) on plasma big ET-1 concentrations in 11 male and 11 female healthy subjects. Data analysis revealed that neither sex nor body position influenced plasma big ET-1 concentrations. This assay should thus permit the detection of possible variations in plasma concentrations of big ET-1 in certain pathologies and, in association with ET-1 assay, make possible in vitro study of endothelin-converting enzyme activity in cell models. Such studies could clarify the physiological and clinical roles of this family of peptides.

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

PubMed Central

Turunen, H J

1983-01-01

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis. PMID:6345574

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

PubMed

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

2010-08-01

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

A rapid magnetic particle-based enzyme immunoassay for human cytomegalovirus glycoprotein B quantification.

PubMed

Pires, F; Arcos-Martinez, M Julia; Dias-Cabral, A Cristina; Vidal, Juan C; Castillo, Juan R

2018-04-17

Human cytomegalovirus (HCMV) is a herpes virus that can cause severe infections. Still, the available methods for its diagnostic have the main disadvantage of requiring long time to be performed. In this work, a simple magnetic particle-based enzyme immunoassay (mpEIA) for the quantification of glycoprotein B of Human cytomegalovirus (gB-HCMV) in urine samples is proposed. The immunosensor scheme is based on the analyte protein gB-HCMV sandwiched between a primary monoclonal antibody, (MBs-PrG-mAb1), and a secondary anti-gB-HCMV antibody labelled with Horseradish peroxidase (Ab2-HRP) to allow spectrophotometric detection. The mpEIA analytical performance was tested in urine samples, showing a linear dependence between gB-HCMV concentration and the absorbance signal at 450 nm in a range of concentrations from 90 to 700 pg mL -1 . The calculated detection limits for gB-HCMV were 90 ± 2 pg mL -1 and the RSD was about 6.7% in urine samples. The immunosensor showed good selectivity against other viruses from Herpesviridae family, namely varicella zoster and Epstein Barr viruses. The recoveries of spiked human urine samples at 0.30-0.50 ng mL -1 concentration levels of gB-HCMV ranged between 91 to 105%. The proposed mpEIA method was validated following the guidelines of the European Medicines Agency (EMEA-2014), and allows rapid, successful and easy quantification of gB-HCMV in urine samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Laser fluorometric analysis of plants for uranium exploration

USGS Publications Warehouse

Harms, T.F.; Ward, F.N.; Erdman, J.A.

1981-01-01

A preliminary test of biogeochemical exploration for locating uranium occurrences in the Marfa Basin, Texas, was conducted in 1978. Only 6 of 74 plant samples (mostly catclaw mimosa, Mimosa biuncifera) contained uranium in amounts above the detection limit (0.4 ppm in the ash) of the conventional fluorometric method. The samples were then analyzed using a Scintrex UA-3 uranium analyzer* * Use of trade names in this paper is for descriptive purposes only and does not constitute endorsement by the U.S. Geological Survey. - an instrument designed for direct analysis of uranium in water, and which can be conveniently used in a mobile field laboratory. The detection limit for uranium in plant ash (0.05 ppm) by this method is almost an order of magnitude lower than with the fluorometric conventional method. Only 1 of the 74 samples contained uranium below the detection limit of the new method. Accuracy and precision were determined to be satisfactory. Samples of plants growing on mineralized soils and nonmineralized soils show a 15-fold difference in uranium content; whereas the soils themselves (analyzed by delayed neutron activation analysis) show only a 4-fold difference. The method involves acid digestion of ashed tissue, extraction of uranium into ethyl acetate, destruction of the ethyl acetate, dissolution of the residue in 0.005% nitric acid, and measurement. ?? 1981.

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

PubMed

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

2015-02-15

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. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2011-07-01

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Automated differential fluorometric analysis of norepinephrine and epinephrine in blood plasma and urine.

DOT National Transportation Integrated Search

1971-04-01

An automated fluorometric trihydroxyindole procedure is described for the measurement of norepinephrine (NE) and epinephrine (E) in blood plasma or urine. The method employs conventional techniques for isolation of the catecholamines by alumina colum...

Immunoassays in Biotechnology

EPA Science Inventory

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

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

USGS Publications Warehouse

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

1987-01-01

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

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...

Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens

PubMed Central

Tanaka, M.; Nakayama, H.; Sagiyama, K.; Haraoka, M.; Yoshida, H.; Hagiwara, T.; Akazawa, K.; Naito, S.

2000-01-01

Aims—To compare the performance of a new generation dual amplified enzyme immunoassay (EIA) with a molecular method for the diagnosis of Chlamydia trachomatis, using a range of urogenital samples, and to assess the reliability of testing self collected vaginal specimens compared with clinician collected vaginal specimens. Methods—Two population groups were tested. For the first population group, first void urine samples were collected from 193 male patients with urethritis, and endocervical swabs were collected from 187 high risk commercial sex workers. All urine and endocervical specimens were tested by a conventional assay (IDEIA chlamydia), a new generation amplified immunoassay (IDEIA PCE chlamydia), and the Amplicor polymerase chain reaction (PCR). Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. For the second population group, four swab specimens, including one patient obtained vaginal swab, two clinician obtained endocervical swabs, and one clinician obtained vaginal swab, were collected from 91 high risk sex workers. Self collected and clinician collected vaginal swabs were tested by IDEIA PCE chlamydia. Clinician obtained endocervical swabs were assayed by IDEIA PCE chlamydia and Amplicor PCR. Results—The performance of the IDEIA PCE chlamydia test was comparable to that of the Amplicor PCR test when male urine and female endocervical swab specimens were analysed. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. The positivity rates for patient collected vaginal specimens and clinician collected vaginal specimens by IDEIA PCE were 25.2% and 23.1%, respectively, whereas those for clinician collected endocervical swabs by PCR and IDEIA PCE were both 27

[DIAGNOSTIC VALUE OF COMBINED USE OF COMBINED METHOD OF ENZYME IMMUNOASSAY AND POLYMERASE CHAIN REACTION TO DETECT OF INTRAUTERINE FETAL INFECTION BY PARVOVIRUS B19].

PubMed

Bondarenko, N P; Lakatosh, V P; Lakatosh, P V; Malanchuk, O B; Poladich, I V

2015-01-01

The combined method of diagnosis parvovirus infection during pregnancy by maternal serum enzyme immunoassay and deoxyribonucleic acid isolation parvovirus B19 polymerase chain reaction in amnniotic fluid and fetal cord blood newborns, can diagnose vertical transmission and anticipate a negative effect on the fetus parvovirus. Lack of maternal IgM antibodies in serum due to parvovirus seroconversion during pregnancy does not exclude the persistence of the virus in the fetus. To analyze the diagnostic value of the method for determining the LHP parvovirus B19 DNA in the amniotic fluid, umbilical cord blood of newborns to determine vertical transmission of parvovirus infection when infected mothers B19 during pregnancy.

Stabilization of penicillinase-hapten conjugate for enzyme immunoassay.

PubMed

Omidfar, K; Rasaee, Mohammad J; Zaraee, Ali B; Amir, M Pour; Rahbarizadeh, F

2002-01-01

The influence of various additives, such as organic solvents, polyhydric alcohols, salts, polymers, and cross-linker, on the stability and storage ability of penicillinase-morphine conjugate was studied in liquid and solid (freeze dried) states. The results of these experiments showed that using low concentrations of CaCl2 (0.1-0.2%) could stabilize enzyme activity in both states for more than seven months. The immunoreactivity of antigen toward the antibody did not change significantly. However, a cross-linker such as glutaraldehyde and various additives such as dimethylsulfoxide, glycerol, polyethylene glycol, gelatin, dextran, ammonium sulfate, lactose, and sucrose did not have any effect on stability. In addition, it was found that the presence of lactose and sucrose in the lyophilization procedure gives a significant amount of protection to the enzyme, which could last for a period of seven months and preserve almost 95% of the enzyme activity, as well as immunoreactivity of the tracer molecule.

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

PubMed

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

2014-01-01

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. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reliability of the cloned-enzyme donor immunoassay (CEDIA) for cocaine in human serum in the range between the detection limit and the cut-off.

PubMed

Schütz, Harald; Auch, Jürgen; Erdmann, Freidoon; Weiler, Günter; Verhoff, Marcel A

2006-01-01

Immunoassays are used worldwide for the rapid screening of drugs. Despite the fact that they are highly valuable tools for the testing of legal and illicit drugs, there is a non-negligable risk of false-positive and false-negative findings and many pitfalls must be taken into account when using these tests in an uncritical manner and without valid confirmation procedures. In order to check the correlation between cloned-enzyme donor immunoassay (CEDIA) readings and exact determined gas chromatography/mass spectrometry (GC/MS) values for benzoylecgonine, a total of 472 serum samples was measured with an immunoassay (CEDIA) as well as GC/MS. As a result, it was shown that in the lower area of concentration, up to approx. 300 ng equ. benzoylecgonine/ml, there is a semiquantitative useful correlation. With higher concentrations, the values shift more and more apart and many runaways are visible. Generally the use of tests for materials other than those declared is excluded from the manufacturer's warranty and is at the user's own risk, but the authors found that many immunochemical tests with a certification only for the use in urine are well suited to the screening of serum samples, and even low readings could be confirmed by GC/MS. With blood or serum as sample, it could be regularly observed that generally very small readings could not only be confirmed with mass spectrometry but could in most cases also be quantified. This was the key factor in determining the rate of confirmation in 495 cases. In total, an incredibly high reliability for the CEDIA cocaine test (via benzoylecgonine) was shown up to the area of detection (detection limit), far below the cut-off. These results confirm the extraordinary sensitivity and specificity of this test also for blood or serum as sample material to be tested.

[Fundamental evaluation of apolipoprotein B-48 by chemiluminescence enzyme immunoassay--identification of apolipoprotein B-48 with immunoblotting].

PubMed

Sato, Itsuko; Fujioka, Yoshio; Hayashi, Fujio; Mukai, Masahiko; Kawano, Seiji; Ishikawa, Yuichi; Yamashita, Shizuya; Kumagai, Shunichi

2007-06-01

Apolipoprotein B-48 (apo B-48) is a constituent of chylomicrons and chylomicron remnants, and its fasting concentration has been reported to be a marker of postprandial hyperlipidemia, which is thought to be a risk factor of atherosclerosis. We evaluated the serum apo B-48 concentrations by chemiluminescence enzyme immunoassay (CLEIA), which was recently introduced as Lumipulse f fully automated immunosaasy analyzer by Fujirebio Inc (Tokyo, Japan), and performed immunoblotting on agarose gel electrophoresis with anti-apo B-48 antibody. Apo B-48 assay was intra-assay reproducible (CVs: 1.9-3.1%) and inter-assay reproducible (CVs: 2.2-4.4%). The assay range for apo B-48 was from 0.2 to 40.0 microg/ml. The effects of interfering substances such as free/conjugated birirubin, hemoglobin, Intrafat, ascorbic acid and rheumatoid factor were negligible. For storage, it was preferable to freeze, and to avoid frozen-thaw process as much as possible. Anti-apo B-48 antibody was reactive over a wide range from origin to the position of very-low-density lipoproteins in immunoblotting after agarose gel electrophoresis. Apo B-48 measurement by CLEIA was feasible to clinical use for the assessment of lipoprotein metabolism.

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

PubMed

Wei, Hui; Wang, Erkang

2013-07-21

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).

Automatic miniaturized fluorometric flow system for chemical and toxicological control of glibenclamide.

PubMed

Ribeiro, David S M; Prior, João A V; Taveira, Christian J M; Mendes, José M A F S; Santos, João L M

2011-06-15

In this work, and for the first time, it was developed an automatic and fast screening miniaturized flow system for the toxicological control of glibenclamide in beverages, with application in forensic laboratory investigations, and also, for the chemical control of commercially available pharmaceutical formulations. The automatic system exploited the multipumping flow (MPFS) concept and allowed the implementation of a new glibenclamide determination method based on the fluorometric monitoring of the drug in acidic medium (λ(ex)=301 nm; λ(em)=404 nm), in the presence of an anionic surfactant (SDS), promoting an organized micellar medium to enhance the fluorometric measurements. The developed approach assured good recoveries in the analysis of five spiked alcoholic beverages. Additionally, a good agreement was verified when comparing the results obtained in the determination of glibenclamide in five commercial pharmaceutical formulations by the proposed method and by the pharmacopoeia reference procedure. Copyright © 2011 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hua; Wang, Jun; Choi, Daiwon

2009-03-01

A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulationmore » 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.« less

Simplified urinary immunoassay for 2,4-D: validation and exposure assessment.

PubMed

Lyubimov, A V; Garry, V F; Carlson, R E; Barr, D B; Baker, S E

2000-08-01

Urinary monitoring of exposed workers by either analytic chemical methods or radioimmunoassay suggests that urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) exceeding 30 ppb are indicative of occupational exposure. However, the current methods do not lend themselves to clinical laboratory use in the rural medical setting. The major goal of this project was to provide medical practitioners who care for members of the agricultural community with a cost-efficient way to conduct exposure assessment. This project used a direct 2,4-D enzyme immunoassay (EIA) and measurement of the ratio between 2,4-D-spiked and non-spiked samples of the same urine to quantify 2,4-D levels. This simplified approach minimizes the effects of non-specific interfering substances in urine and eliminates the need for sample extraction and clean-up. Possible urine co-contaminants (2,4-dichlorophenol and 2,5-dichlorophenol) do not significantly interfere with this immunoassay. Twenty-two forest pesticide applicators who apply and use chlorophenoxy herbicides in their work and 14 comparable control subjects were studied to validate the assay in the occupational setting. Coded urine specimens were examined for levels of 2,4-D by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and compared with immunoassay results from the same specimens. A correlation coefficient of r = 0.982 with a P value of .0001 for a plot of HPLC-MS/MS versus immunoassay demonstrated that the results from these methods were comparable over urinary dose levels ranging from not detectable (<19 ppb) to 1700 ppb 2,4-D, as determined by immunoassay.

Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays

PubMed Central

Chen, Hui; Hagström, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.

2016-01-01

In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

Enzyme immunoassay of Chlamydia in birds.

PubMed

Ruppanner, R; Behymer, D E; DeLong, W J; Franti, C E; Schulz, T

1984-01-01

Serum samples from 192 free-living birds (27 species) were tested for antibodies against Chlamydia using the enzyme-linked immunosorbent assay (ELISA); 97 (51%) were seropositive. The highest antibody prevalence was among pheasants (96%), ducks (88%), and blackbirds (86%). None of 41 starlings tested were seropositive. The serotesting of 42 confined pheasants indicated 100% exposure to the organism. The ELISA is a sensitive, rapid serologic method that can be of epidemiologic and diagnostic value for detecting exposure to Chlamydia. The ELISA could also be used for mass-screening of pet birds where chlamydiosis may be considered a potential public health hazard.

Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations

PubMed Central

Schwab, Karen; Lauber, Jennifer; Hesse, Friedemann

2016-01-01

The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle® T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso® B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L−1 and 0.08 g·L−1, as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10−4 µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min−1·µg−1) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems. PMID:28952595

Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

PubMed

Gaafar, Maha R

2011-08-01

This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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

PubMed Central

Harris, Michael J.; Dugger, William M.

1986-01-01

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

Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yinfa, Ma.

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will bemore » described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.« less

Bupropion interference with immunoassays for amphetamines and LSD.

PubMed

Vidal, Christian; Skripuletz, Thomas

2007-06-01

A 50-year-old male patient suddenly had lost consciousness, although he had previously been healthy. On arrival at hospital seizures arose. The authors investigated a urine sample of the patient, and performed toxicological drug screening with immunochemical Cloned Enzyme Donor Immunoassay (CEDIA) assays. Positive findings for amphetamines and LSD could not be confirmed. Using gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS), the authors identified bupropion, a drug used to aid in smoking cessation, as the interfering compound, which may cause false-positive results for amphetamines and LSD using the CEDIA assays.

A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.

PubMed

Marcella, Aaron M; Barb, Adam W

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured K M values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a k cat of 7.4-9.0 s -1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed K M values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a k cat of 2100-2600 s -1 for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

Materials for Microfluidic Immunoassays: A Review.

PubMed

Mou, Lei; Jiang, Xingyu

2017-08-01

Conventional immunoassays suffer from at least one of these following limitations: long processing time, high costs, poor user-friendliness, technical complexity, poor sensitivity and specificity. Microfluidics, a technology characterized by the engineered manipulation of fluids in channels with characteristic lengthscale of tens of micrometers, has shown considerable promise for improving immunoassays that could overcome these limitations in medical diagnostics and biology research. The combination of microfluidics and immunoassay can detect biomarkers with faster assay time, reduced volumes of reagents, lower power requirements, and higher levels of integration and automation compared to traditional approaches. This review focuses on the materials-related aspects of the recent advances in microfluidics-based immunoassays for point-of-care (POC) diagnostics of biomarkers. We compare the materials for microfluidic chips fabrication in five aspects: fabrication, integration, function, modification and cost, and describe their advantages and drawbacks. In addition, we review materials for modifying antibodies to improve the performance of the reaction of immunoassay. We also review the state of the art in microfluidic immunoassays POC platforms, from the laboratory to routine clinical practice, and also commercial products in the market. Finally, we discuss the current challenges and future developments in microfluidic immunoassays. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Performance of a Limiting-Antigen Avidity Enzyme Immunoassay for Cross-Sectional Estimation of HIV Incidence in the United States

PubMed Central

Konikoff, Jacob; Brookmeyer, Ron; Longosz, Andrew F.; Cousins, Matthew M.; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Kirk, Gregory D.; Moore, Richard D.; Mehta, Shruti H.; Margolick, Joseph B.; Brown, Joelle; Mayer, Kenneth H.; Koblin, Beryl A.; Justman, Jessica E.; Hodder, Sally L.; Quinn, Thomas C.; Eshleman, Susan H.; Laeyendecker, Oliver

2013-01-01

Background A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. Methods and Findings Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. Conclusions The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence

Isotachophoresis-Based Surface Immunoassay.

PubMed

Paratore, Federico; Zeidman Kalman, Tal; Rosenfeld, Tally; Kaigala, Govind V; Bercovici, Moran

2017-07-18

In the absence of amplification methods for proteins, the immune-detection of low-abundance proteins using antibodies is fundamentally limited by binding kinetic rates. Here, we present a new class of surface-based immunoassays in which protein-antibody reaction is accelerated by isotachophoresis (ITP). We demonstrate the use of ITP to preconcentrate and deliver target proteins to a surface decorated with specific antibodies, where effective utilization of the focused sample is achieved by modulating the driving electric field (stop-and-diffuse ITP mode) or applying a counter flow that opposes the ITP motion (counterflow ITP mode). Using enhanced green fluorescent protein (EGFP) as a model protein, we carry out an experimental optimization of the ITP-based immunoassay and demonstrate a 1300-fold improvement in limit of detection compared to a standard immunoassay, in a 6 min protein-antibody reaction. We discuss the design of buffer chemistries for other protein systems and, in concert with experiments, provide full analytical solutions for the two operation modes, elucidating the interplay between reaction, diffusion, and accumulation time scales and enabling the prediction and design of future immunoassays.

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

PubMed

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

2011-01-01

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 μg 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 μg 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.

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 ...

Fluorometric immunoassay for human serum albumin based on its inhibitory effect on the immunoaggregation of quantum dots with silver nanoparticles

NASA Astrophysics Data System (ADS)

Marukhyan, Seda S.; Gasparyan, Vardan K.

2017-02-01

Quantitative determination of HSA was conducted by competitive immunoassay. Inhibition of aggregation of antibody conjugated quantum dots (QD) with albumin conjugated silver nanoparticles (AgNPs) in the presence of HSA was conducted. If antibody-loaded CdSe QDs aggregate with HSA-coated silver nanoparticles the distance between the two kinds of nanoparticles will be reduced enough to cause fluorescence resonance energy transfer (FRET). In this case the yellow fluorescence of the Ab-QDs is quenched. However if HSA (antigen) is added to the Ab-QDs their surface will be blocked and they cannot aggregate any longer with the HSA-AgNPs. Hence, fluorescence will not be quenched. The drop of the intensity of fluorescence (peaking at 570 nm) is inversely correlated with the concentration of HSA in the sample. The method allows to determine HSA in the 30-600 ng·mL-1 concentration range.

Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

PubMed

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

2010-10-27

A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

[A system review on the application of BED-capture enzyme immunoassay in detecting new HIV-1 infection].

PubMed

Tao, Jun; Zhao, Jing; Liu, Yong; Meng, Ling-zhang; Yu, Shi-cheng; Jiang, Yan; Xiao, Yao

2011-02-01

To study whether BED-capture enzyme immunoassay (CEIA) is feasible to be used in wide-ranging population, we collect papers and conference abstracts related to BED-CEIA and HIV-1 incidence. 10 papers are included for the discussion, regarding the concordance between the estimated HIV-1 incidence from BED-CEIA and the results from a cohort studies; and 11 papers are selected to discuss the related misclassification on the estimation of HIV-1 incidence. Concordance between the two sets is related to the districts and design of research. Results from Africa are not so satisfactory, but researches, those BED-CEIA samples are collected during the follow-up of the cohort study, have shown better outcomes than other ones. There are totally 7303 samples of LTI (long-term infections) collected for analyzing the misclassification of BED-CEIA. 432 LTI are misclassified as new infections, making the raw rate of misclassification as 5.9% with 95% confidential interval between 5.36 and 6.44. Data from systematic review shows that the BED-CEIA's misclassification rate relates to the count of CD4(+)T lymphocytes and time after the infection but has no relation to the classification of sub-populations (female sexual workers and intravenous drug users in China) and districts (China and Africa). Our results reveal that the misclassification is relevant to the immune-status of the infected persons.

BODIPY-based fluorometric sensor array for the highly sensitive identification of heavy-metal ions.

PubMed

Niu, Li-Ya; Li, Hui; Feng, Liang; Guan, Ying-Shi; Chen, Yu-Zhe; Duan, Chun-Feng; Wu, Li-Zhu; Guan, Ya-Feng; Tung, Chen-Ho; Yang, Qing-Zheng

2013-05-02

A BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based fluorometric sensor array has been developed for the highly sensitive detection of eight heavy-metal ions at micromolar concentration. The di-2-picolyamine (DPA) derivatives combine high affinities for a variety of heavy-metal ions with the capacity to perturb the fluorescence properties of BODIPY, making them perfectly suitable for the design of fluorometric sensor arrays for heavy-metal ions. 12 cross-reactive BODIPY fluorescent indicators provide facile identification of the heavy-metal ions using a standard chemometric approach (hierarchical clustering analysis); no misclassifications were found over 45 trials. Clear differentiation among heavy-metal ions as a function of concentration was also achieved, even down to 10(-7)M. A semi-quantitative interpolation of the heavy-metal concentration is obtained by comparing the total Euclidean distance of the measurement with a set of known concentrations in the library. Copyright © 2013 Elsevier B.V. All rights reserved.

Application of a fluorometric microplate algal toxicity assay for riverine periphytic algal species.

PubMed

Nagai, Takashi; Taya, Kiyoshi; Annoh, Hirochica; Ishihara, Satoru

2013-08-01

Although riverine periphytic algae attached to riverbed gravel are dominant species in flowing rivers, there is limited toxicity data on them because of the difficulty in cell culture and assays. Moreover, it is well known that sensitivity to pesticides differ markedly among species, and therefore the toxicity data for multiple species need to be efficiently obtained. In this study, we investigated the use of fluorometric microplate toxicity assay for testing periphytic algal species. We selected five candidate test algal species Desmodesmus subspicatus, Achnanthidium minutissimum, Navicula pelliculosa, Nitzschia palea, and Pseudanabaena galeata. The selected species are dominant in the river, include a wide range of taxon, and represent actual species composition. Other additional species were also used to compare the sensitivity and suitability of the microplate assay. A 96-well microplate was used as a test chamber and algal growth was measured by in-vivo fluorescence. Assay conditions using microplate and fluorometric measurement were established, and sensitivities of 3,5-dichlorophenol as a reference substance were assayed. The 50 percent effect concentrations (EC50s) obtained by fluorometric microplate assay and those obtained by conventional Erlenmeyer flask assay conducted in this study were consistent. Moreover, the EC50 values of 3,5-dichlorophenol were within the reported confidence intervals in literature. These results supported the validity of our microplate assay. Species sensitivity distribution (SSD) analysis was conducted using the EC50s of five species. The SSD was found to be similar to the SSD obtained using additional tested species, suggesting that SSD using the five species largely represents algal sensitivity. Our results provide a useful and efficient method for high-tier probabilistic ecological risk assessment of pesticides. Copyright © 2013 Elsevier Inc. All rights reserved.

Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

NASA Astrophysics Data System (ADS)

Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

2017-02-01

An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

PubMed

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

2015-11-01

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

Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

PubMed

Ogata, Norio

2006-04-01

The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated.

Fluorometric Method for Determining the Efficiency of Spun-Glass Air Filtration Media

PubMed Central

Sullivan, James F.; Songer, Joseph R.; Mathis, Raymond G.

1967-01-01

The procedures and equipment needed to measure filtration efficiency by means of fluorescent aerosols are described. The filtration efficiency of individual lots of spun-glass air filtration medium or of entire air filtration systems employing such media was determined. Data relating to the comparative evaluation of spun-glass filter media by means of the fluorometric method described, as well as by conventional biological procedures, are presented. PMID:6031433

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

PubMed

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

2004-01-01

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

Evaluation of Correlation between Pretest Probability for Clostridium difficile Infection and Clostridium difficile Enzyme Immunoassay Results

PubMed Central

Reske, Kimberly A.; Hink, Tiffany; Dubberke, Erik R.

2016-01-01

ABSTRACT The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI. PMID:27927930

Fluorometric determination of nitrite in cured meats.

PubMed

Coppola, E D; Wickroski, A F; Hanna, J G

1975-05-01

An indirect fluorometric method for determining sodium nitrite in meat products is presented. The extracted sodium nitrite is consumed in a diazotization reaction with a measured excess of sulfanilic acid. Fluorescamine, which acts selectively with primary amines such as sulfanilic acid, is a fluorogenic reagent for the excess amine. The amine consumed, calculated by difference from the total originally present, is directly related to the sodium nitrite content of the sample. Interferences from amino acids and soluble proteins in the meat extract are eliminated by judicious use of a secondary peak in the fluorescence spectra (436 nm excitation, 495 nm fluorescence) combined with measurement at low pH (3.30). The recoveries of sodium nitrite ranged from 83.2 to 99.6% with an average of 93.4 and a standard deviation of +/- 5.28% for 11 determinations.

Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.

PubMed

Gross, Madeleine; Curtui, Valeriu; Usleber, Ewald

2018-05-01

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

Fluorometric method for inorganic pyrophosphatase activity detection and inhibitor screening based on click chemistry.

PubMed

Xu, Kefeng; Chen, Zhonghui; Zhou, Ling; Zheng, Ou; Wu, Xiaoping; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

2015-01-06

A fluorometric method for pyrophosphatase (PPase) activity detection was developed based on click chemistry. Cu(II) can coordinate with pyrophosphate (PPi), the addition of pyrophosphatase (PPase) into the above system can destroy the coordinate compound because PPase catalyzes the hydrolysis of PPi into inorganic phosphate and produces free Cu(II), and free Cu(II) can be reduced by sodium ascorbate (SA) to form Cu(I), which in turn initiates the ligating reaction between nonfluorescent 3-azidocoumarins and terminal alkynes to produce a highly fluorescent triazole complex, based on which, a simple and sensitive turn on fluorometric method for PPase can be developed. The fluorescence intensity of the system has a linear relationship with the logarithm of the PPase concentration in the range of 0.5 and 10 mU with a detection limit down to 0.2 mU (S/N = 3). This method is cost-effective and convenient without any labels or complicated operations. The proposed system was applied to screen the potential PPase inhibitor with high efficiency. The proposed method can be applied to diagnosis of PPase-related diseases.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

PubMed

Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

2015-09-01

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Detection of alpha-fetoprotein in magnetic immunoassay of thin channels using biofunctional nanoparticles

NASA Astrophysics Data System (ADS)

Tsai, H. Y.; Gao, B. Z.; Yang, S. F.; Li, C. S.; Fuh, C. Bor

2014-01-01

This paper presents the use of fluorescent biofunctional nanoparticles (10-30 nm) to detect alpha-fetoprotein (AFP) in a thin-channel magnetic immunoassay. We used an AFP model biomarker and s-shaped deposition zones to test the proposed detection method. The results show that the detection using fluorescent biofunctional nanoparticle has a higher throughput than that of functional microparticle used in previous experiments on affinity reactions. The proposed method takes about 3 min (versus 150 min of previous method) to detect 100 samples. The proposed method is useful for screening biomarkers in clinical applications, and can reduce the run time for sandwich immunoassays to less than 20 min. The detection limits (0.06 pg/ml) and linear ranges (0.068 pg/ml-0.68 ng/ml) of AFP using fluorescent biofunctional nanoparticles are the same as those of using functional microparticles within experimental errors. This detection limit is substantially lower and the linear range is considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other methods in sandwich immunoassay methods. The differences between this method and an ELISA in AFP measurements of serum samples were less than 12 %. The proposed method provides simple, fast, and sensitive detection with a high throughput for biomarkers.

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin.

PubMed

Furzer, Gordon S; Veldhuis, Linda; Hall, J Christopher

2006-02-08

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.

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

NASA Astrophysics Data System (ADS)

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

1997-02-01

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

Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

PubMed Central

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

2014-01-01

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

Development of an incurred cornbread model for gluten detection by immunoassays.

PubMed

Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

2013-12-11

Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

Hydrogel nanoparticle based immunoassay

DOEpatents

Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

2015-04-21

An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

PubMed

Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

2016-11-01

A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Industrial Fungal Enzymes: An Occupational Allergen Perspective

PubMed Central

Green, Brett J.; Beezhold, Donald H.

2011-01-01

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

Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

PubMed Central

Musher, Benjamin; Fredricks, David; Leisenring, Wendy; Balajee, S. Arunmozhi; Smith, Caitlin; Marr, Kieren A.

2004-01-01

Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillus species from BAL samples of patients with proven and probable IPA (case patients; n = 49) and without IPA (control patients; n = 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures. PMID:15583275

Evaluation of Correlation between Pretest Probability for Clostridium difficile Infection and Clostridium difficile Enzyme Immunoassay Results.

PubMed

Kwon, Jennie H; Reske, Kimberly A; Hink, Tiffany; Burnham, C A; Dubberke, Erik R

2017-02-01

The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI. Copyright © 2017 American Society for Microbiology.

Fluorometric determination of histamine in cheese.

PubMed

Chambers, T L; Staruszkiewicz, W F

1978-09-01

Thirty-one samples of cheese obtained from retail outlets were analyzed for histamine, using an official AOAC fluorometric method. The types of cheese analyzed and the ranges of histamine found were: colby, 0.3--2.8; camembert, 0.4--4.2; cheddar, 1.2--5.8; gouda, 1.3--2.4; provolone, 2.0--23.5; roquefort, 1.0--16.8; mozzarella 1.6--5.0; and swiss, 0.4--250 mg histamine/100 g. Ten of the 12 samples of swiss cheese contained less than 16 mg histamine/100 g. The remaining 2 samples which contained 116 and 250 mg histamine/100 g were judged organoleptically to be of poor quality. An investigation of one processing facility showed that the production of histamine in swiss cheese may have been a result of a hydrogen peroxide/low temperature treatment of the milk supply. Recovery of histamine added to methanol extracts of cheese ranged from 93 to 105%. Histamine content was confirmed by high pressure liquid chromatographic analysis of the methanol extracts.

Fluorometric determination of zirconium in minerals

USGS Publications Warehouse

Alford, W.C.; Shapiro, L.; White, C.E.

1951-01-01

The increasing use of zirconium in alloys and in the ceramics industry has created renewed interest in methods for its determination. It is a common constituent of many minerals, but is usually present in very small amounts. Published methods tend to be tedious, time-consuming, and uncertain as to accuracy. A new fluorometric procedure, which overcomes these objections to a large extent, is based on the blue fluorescence given by zirconium and flavonol in sulfuric acid solution. Hafnium is the only element that interferes. The sample is fused with borax glass and sodium carbonate and extracted with water. The residue is dissolved in sulfuric acid, made alkaline with sodium hydroxide to separate aluminum, and filtered. The precipitate is dissolved in sulfuric acid and electrolysed in a Melaven cell to remove iron. Flavonol is then added and the fluorescence intensity is measured with a photo-fluorometer. Analysis of seven standard mineral samples shows excellent results. The method is especially useful for minerals containing less than 0.25% zirconium oxide.

[Comparative sensitivity of different solid-phase immunoassays for detecting HBsAg and anti-HCV in blood].

PubMed

León, P; López, J A; Domingo, C J; Echevarría, J M

1992-10-01

The sensitivities of seven methods of enzyme-immunoassay for HBsAg detection, as well as of twelve immunoassays for detecting antibody to HCV, were compared, in an attempt to evaluate the need for an official technical validation of such methods prior to its commercialization in Spain. Significant differences were seen for the sensitivities of these methods, which may influence the proficiency of blood bank screening for HBsAg and anti-HCV for preventing cases of post-transfusional viral hepatitis. As a conclusion, it is recommended to establish legal regulations for pre-marketing validation of such assays in Spain, as well as to take the results obtained in these evaluation studies as the basis for selecting those tests which may be more convenient for screening purposes at the blood banks.

Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

PubMed

Cortina, María E; Novak, Analía; Melli, Luciano J; Elena, Sebastián; Corbera, Natalia; Romero, Juan E; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

2017-09-01

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans. Copyright © 2017 Elsevier B.V. All rights reserved.

A selective electromembrane extraction of uranium (VI) prior to its fluorometric determination in water.

PubMed

Davarani, Saied Saeed Hosseiny; Moazami, Hamid Reza; Keshtkar, Ali Reza; Banitaba, Mohammad Hossein; Nojavan, Saeed

2013-06-14

A novel method for the selective electromembrane extraction (EME) of U(6+) prior to fluorometric determination has been proposed. The effect of extraction conditions including supported liquid membrane (SLM) composition, extraction time and extraction voltage were investigated. An SLM composition of 1% di-2-ethyl hexyl phosphonic acid in nitrophenyl octyl ether (NPOE) showed good selectivity, recovery and enrichment factor. The best performance was achieved at an extraction potential of 80 volts and an extraction time of 14 minutes Under the optimized conditions, a linear range from 1 to 1000 ng mL(-1) and LOD of 0.1 ng mL(-1) were obtained for the determination of U(6+). The EME method showed good performance in sample cleanup and the reduction of the interfering effects of Mn(2+), Zn(2+), Cd(2+), Ni(2+), Fe(3+), Co(2+), Cu(2+), Cl(-) and PO4(3-) ions during fluorometric determination of uranium in real water samples. The recoveries above 54% and enrichment factors above 64.7 were obtained by the proposed method for real sample analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

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. ...

Fluorometric method for the determination of gas-phase hydrogen peroxide

NASA Technical Reports Server (NTRS)

Kok, Gregory L.; Lazrus, Allan L.

1986-01-01

The fluorometric gas-phase hydrogen peroxide procedure is based on the technique used by Lazrus et. al. for the determination of H2O2 in the liquid phase. The analytical method utilizes the reaction of H2O2 with horseradish peroxidase and p-hydroxphenylacetic acid (POPHA) to form the fluorescent dimer of POPHA. The analytical reaction responds stoichiometrically to both H2O2 and some organic hydroperoxides. To discriminate H2O2 from organic hydroperoxides, catalase is used to preferentially destroy H2O2. Using a dual-channel flow system the H2O2 concentration is determined by difference.

Protein adsorption in microengraving immunoassays.

PubMed

Song, Qing

2015-10-16

Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

Protein Adsorption in Microengraving Immunoassays

PubMed Central

Song, Qing

2015-01-01

Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

PubMed

Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

2007-03-01

To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

NASA Astrophysics Data System (ADS)

Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

2018-04-01

In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

Sensitive detection of respiratory syncytial virus based on a dual signal amplified plasmonic enzyme-linked immunosorbent assay.

PubMed

Zhan, Lei; Wu, Wen Bi; Yang, Lin; Huang, Cheng Zhi

2017-04-15

The timely detection of infectious pathogen is critical in clinical early diagnosis and treatment of infectious diseases. Plasmonic enzyme-linked immunosorbent assay (ELISA), by means of enzyme-mediated growth or aggregation of AuNPs, has received considerable attention because it allows a naked-eye detection of target in very low numbers. In this work, a dual-signal amplified plasmonic ELISA combined the high loading capacity of magnetic beads with the establishing stimulation effect of zinc ion has been developed to detect RSV as a model pathogen based on alkaline phosphatase-triggered dispersion of aggregated AuNPs. In ideal conditions, the proposed immunoassay can conveniently distinguish the concentration of RSV in a range of 0.1-30 pg/mL. In addition, the limit of detection of RSV of this immunoassay exceeds that of conventional ELISA by about 50 times. The high sensitivity makes this approach a good alternative to existing colorimetric immunoassays for pathogen detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid micromotor-based naked-eye immunoassay.

PubMed

de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

2017-05-15

A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes. Copyright © 2017 Elsevier B.V. All rights reserved.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

PubMed

Sista, Ramakrishna S; Eckhardt, Allen E; Wang, Tong; Graham, Carrie; Rouse, Jeremy L; Norton, Scott M; Srinivasan, Vijay; Pollack, Michael G; Tolun, Adviye A; Bali, Deeksha; Millington, David S; Pamula, Vamsee K

2011-10-01

Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Capillary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene glycol) pyrenebutanoate.

PubMed

Horka, Marie; Ruzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

2006-12-15

The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.

Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species.

PubMed

Bienboire-Frosini, Cecile; Chabaud, Camille; Cozzi, Alessandro; Codecasa, Elisa; Pageat, Patrick

2017-01-01

The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although

Usefulness of enzyme immunoassay (EIA) for screening of anti HIV antibodies in urinary specimens: A comparative analysis.

PubMed

Sahni, A K; Nagendra, A; Roy, Partha; Patrikar, S

2014-07-01

Standard HIV testing is done using serum or plasma. FDA approved ELISA to screen urine for IgG antibodies to HIV-1 in 1996. It is a simple, noninvasive test and is appropriate for developing countries where health care personnel may not be professionally trained or where clean needles for drawing blood may not always be available. 436 individuals with high-risk behavior and strong clinical suspicion of HIV infection were screened for IgG antibodies to HIV-1 in urine by ELISA. Urine HIV testing was performed by enzyme immunoassay, at the ongoing Voluntary Confidential Counseling and Testing Center (VCCTC) at a large tertiary care microbiology lab. The individuals enrolled for the study had high-risk exposure to the virus and majorities were from a state with a high incidence of HIV infection. In all individuals, both serum and urine were tested for IgG antibodies to HIV-1. Overall, 135 individuals (30.96%) were HIV-positive, of whom 96 (71%) had never previously tested positive; 87% of those who tested positive received their results, and most were referred for medical care. Sensitivity, specificity and predictive values of HIV-1 urine ELISA test kit were determined. Sensitivity was found to be 89.6%; 95% CI [82.9-94.0], specificity 97.3%; 95% CI [94.6-98.8], positive predictive value 93.8%; 95% CI [87.8-97.1] and negative predictive value 95.4%; 95% CI [92.3-97.4]. Efficiency, sensitivity, and specificity of the urine-based screening for HIV-1 test kits were excellent as compared to the reference test.

Development of an enzyme immunoassay for detection of antibodies against Coccidioides in dogs and other mammalian species.

PubMed

Chow, Nancy A; Lindsley, Mark D; McCotter, Orion Z; Kangiser, Dave; Wohrle, Ron D; Clifford, Wayne R; Yaglom, Hayley D; Adams, Laura E; Komatsu, Kenneth; Durkin, Michelle M; Baker, Rocky J; Shubitz, Lisa F; Derado, Gordana; Chiller, Tom M; Litvintseva, Anastasia P

2017-01-01

Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species.

Development of an enzyme immunoassay for detection of antibodies against Coccidioides in dogs and other mammalian species

PubMed Central

Lindsley, Mark D.; McCotter, Orion Z.; Kangiser, Dave; Wohrle, Ron D.; Clifford, Wayne R.; Yaglom, Hayley D.; Adams, Laura E.; Komatsu, Kenneth; Durkin, Michelle M.; Baker, Rocky J.; Shubitz, Lisa F.; Derado, Gordana; Chiller, Tom M.; Litvintseva, Anastasia P.

2017-01-01

Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species. PMID:28380017

Basophil activation test compared to skin prick test and fluorescence enzyme immunoassay for aeroallergen-specific Immunoglobulin-E

PubMed Central

2012-01-01

Background Skin prick test (SPT) and fluorescence enzyme immunoassay (FEIA) are widely used for the diagnosis of Immunoglobulin-E (IgE)-mediated allergic disease. Basophil activation test (BAT) could obviate disadvantages of SPT and FEIA. However, it is not known whether BAT gives similar results as SPT or FEIA for aeroallergens. Objectives In this study, we compared the results of SPT, BAT and FEIA for different aeroallergens. Methods We performed BAT, SPT and FEIA in 41 atopic subjects (symptomatic and with positive SPT for at least 1 of 9 common aeroallergens) and 31 non-atopic subjects (asymptomatic and with negative SPT). Results Correlations between SPT and BAT, SPT and FEIA, and BAT and FEIA results were statistically significant but imperfect. Using SPT as the "gold standard", BAT and FEIA were similar in sensitivity. However, BAT had lower specificity than FEIA. False positive (BATposSPTneg) results were frequent in those atopic subjects who were allergic by SPT to a different allergen and rare in non-atopic subjects. The false positivity in atopic subjects was due in part to high levels of serum Total-IgE (T-IgE) levels in atopic individuals that lead to basophil activation upon staining with fluorochrome-labeled anti-IgE. Conclusion As an alternative to SPT in persons allergic to aeroallergens, BAT in its present form is useful for distinguishing atopic from non-atopic persons. However, BAT in its present form is less specific than FEIA when determining the allergen which a patient is allergic to. This is due to IgE staining-induced activation of atopic person's basophils and/or nonspecific hyperreactivity of atopic person's basophils. PMID:22264407

Detection of IgA-class circulating immune complexes (CIC) in sera from patients with IgA nephropathy using a solid-phase anti-C3 Facb enzyme immunoassay (EIA).

PubMed Central

Yagame, M; Tomino, Y; Miura, M; Tanigaki, T; Suga, T; Nomoto, Y; Sakai, H

1987-01-01

The detection of circulating immune complexes (CIC) in sera from patients with IgA nephropathy is described. A solid-phase anti-C3 Facb enzyme immunoassay (EIA) was employed for detection of IgA-, IgG- and IgM-CIC in sera. The C1q-binding enzyme assay was also used for the detection of CIC in sera from these patients and healthy adults. Twenty-two patients with IgA nephropathy, 14 patients with other glomerular diseases and 19 healthy adults were examined by anti-C3 Facb EIA. The levels of IgA-CIC in sera from patients with IgA nephropathy were significantly higher than those in sera from patients with other glomerular diseases and healthy adults. CIC measured by the C1q-binding enzyme assay was detected in some patients with IgA nephropathy. The levels of serum IgA in patients with IgA nephropathy were significantly higher than those in patients with other glomerular diseases and healthy adults. However, there was no significant correlation between the levels of IgA-CIC in sera and those of serum IgA in patients with IgA nephropathy. There was also no significant correlation between the levels of IgA-CIC in sera and the degree of histopathological injuries in the patients. It is concluded that the solid-phase anti-C3 Facb EIA is useful for the detection of IgA-CIC in sera from patients with IgA nephropathy. PMID:3301093

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

PubMed

Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

2018-01-10

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

A redox-mediated chromogenic reaction and application in immunoassay.

PubMed

Yu, Ru-Jia; Ma, Wei; Peng, Mao-Pan; Bai, Zhi-Shan; Long, Yi-Tao

2016-08-31

A novel redox-mediated chromogenic reaction was demonstrated based on the reaction between HAuCl4 and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which generate various color responses from red to green in the resulting solutions. Various redox substance could be used to mediate the reaction and trigger a distinct color response. We established a sensitive hydrogen peroxide colorimetric sensor based on the redox-mediated chromogenic reaction and depicted the application both in detection of enzyme and in an immunoassay. Combining the traditional chromogenic reagent with gold nanoparticles, our assay has the advantage in short response time (within three minutes), high sensitivity (10(-12) g mL(-1) for HBsAg) and stability. Copyright © 2016. Published by Elsevier B.V.

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

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

2015-03-01

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. 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. 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. 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

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

PubMed

Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin

2016-03-01

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

PubMed

Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

2018-03-01

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

Synthesis of New Hydrazone Derivatives for MAO Enzymes Inhibitory Activity.

PubMed

Can, Nafiz Öncü; Osmaniye, Derya; Levent, Serkan; Sağlık, Begüm Nurpelin; İnci, Beril; Ilgın, Sinem; Özkay, Yusuf; Kaplancıklı, Zafer Asım

2017-08-20

In the present work, 14 new 1-substituted-2-phenylhydrazone derivatives were synthesized to evaluate their inhibitory activity against hMAO enzymes. The structures of the newly synthesized hydrazones 2a-2n were characterized by IR, 1H-NMR, 13C-NMR, HR-MS spectroscopic methods. The inhibitory activity of compounds 2a-2n against hMAO-A and hMAO-B enzymes was elucidated by using an in-vitro Amplex Red® reagent assay based on fluorometric methods. According to the activity studies, 2a and 2b were found to be the most active compounds against hMAO-A enzyme, with IC50 values of 0.342 µM and 0.028 µM, respectively. The most active compounds 2a-2b were evaluated by means of enzyme kinetics and docking studies. Moreover, these compounds were subjected to cytotoxicity and genotoxicity tests to establish their preliminary toxicological profiles and were found to be non-cytotoxic and non-genotoxic. Consequently, the findings of this study display the biological importance of compounds 2a, 2b as selective, irreversible and competitive inhibitors of hMAO-A. Docking studies revealed that there is a strong interaction between hMAO-A and the most active compound 2b.

Fluorescence Immunoassay for Cocaine Detection.

PubMed

Nakayama, Hiroshi; Kenjjou, Noriko; Shigetoh, Nobuyuki; Ito, Yuji

2016-04-01

A fluorescence immunoassay (FIA) has been developed for the detection of cocaine using norcocaine labeled with merocyanine dye and a monoclonal antibody specific to cocaine. Using this FIA, the detection range for cocaine was between 20.0 and 1700 μg/L with a limit of detection of 20.0 μg/L. Other cocaine derivatives did not interfere significantly with the detection when using this immunoassay technique with cross-reactivity values of less than 20%. Thus this FIA could be considered a useful tool for the detection of cocaine.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2005-01-01

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

Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

PubMed

Deguchi, Matsuo; Kagita, Masanori; Yoshioka, Nori; Tsukamoto, Hiroko; Takao, Miyuki; Tahara, Kazuko; Maeda, Ikuhiro; Hidaka, Yoh; Yamauchi, Satoshi; Kaneko, Atsushi; Miyakoshi, Hideo; Isomura, Mitsuo

2017-10-06

Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients. © 2017 Wiley Periodicals, Inc.

Enzyme immunoassays for detection of gypsy moth nuclear polyhedrosis virus

Treesearch

Michael Ma

1985-01-01

Enzyme-linked immunosorbent assays (ELISA) were developed for detecting gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (NPV). They were used to detect the presence of NPV in hemoplymph samples collected from infected larvae. The incorporation of hybridoma antibodies with these procedures would make them even more specific for gypsy moth...

Clinical Characteristics and Outcomes of Hematologic Malignancy Patients With Positive Clostridium difficile Toxin Immunoassay Versus Polymerase Chain Reaction Test Results.

PubMed

Ziegler, Matthew; Landsburg, Daniel; Pegues, David; Alby, Kevin; Gilmar, Cheryl; Bink, Kristen; Gorman, Theresa; Moore, Amy; Bonhomme, Brittaney; Omorogbe, Jacqueline; Tango, Dana; Tolomeo, Pam; Han, Jennifer H

2018-04-25

In a cohort of inpatients with hematologic malignancy and positive enzyme immunoassay (EIA) or polymerase chain reaction (PCR) Clostridium difficile tests, we found that clinical characteristics and outcomes were similar between these groups. The method of testing is unlikely to predict infection in this population, and PCR-positive results should be treated with concern.Infect Control Hosp Epidemiol 2018;1-4.

Ultraviolet, Visible, and Fluorescence Spectroscopy

NASA Astrophysics Data System (ADS)

Penner, Michael H.

Spectroscopy in the ultraviolet-visible (UV-Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV-Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli.

PubMed

Birnbaum, S; Bülow, L; Hardy, K; Danielsson, B; Mosbach, K

1986-10-01

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

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

PubMed

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

2015-01-01

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.

Evaluation and reporting of enzyme immunoassay determinations of antibody to herpes simplex virus in sera and cerebrospinal fluid.

PubMed Central

Cremer, N E; Cossen, C K; Hanson, C V; Shell, G R

1982-01-01

Several methods for evaluating and reporting enzyme immunoassay (EIA) determinations of antibody to herpes simplex virus derived from one dilution of single serum samples were studied. An EIA ratio method for serological evidence of current infection from paired serum samples was also evaluated. Optical density (OD) of the reaction at a 1:100 serum dilution and estimated titers obtained by reference of the OD of the serum dilution to a standard curve were compared to the corresponding plotted EIA titer obtained by titration to endpoint. Neither the OD per se nor the estimated titer was completely predictive of the plotted titer (correlation coefficient [r] of 0.824 and 0.817, respectively), and they provided only a semiquantitative measurement of antibody concentration. For an antibody status report, however, OD would be sufficient if related to the cutoff value as an EIA index (OD of sample divided by cutoff OD for positive specimens). The OD of the EIA reaction at a single dilution (1:5) of cerebrospinal fluid, on the other hand, correlated quite well with the titer obtained by titration (r = 0.950). For serological diagnosis of current infection, the OD ratio of convalescence-phase/acute-phase sera was determined at several dilutions. A ratio of greater than or equal to 1.54 was calculated as a reliable index for a significant rise in antibody concentration and compatible with current infection. By determining the convalescent-phase/acute-phase serum ratio at two dilutions, 1:100 and 1:1,000, the EIA ratio method appeared to be a sensitive as or more sensitive than, complement fixation in diagnosing current infection. PMID:6284791

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer.

PubMed

Chang, Yanli; Xu, Jianjun; Zhang, Qingyun

2017-10-01

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer.

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer

PubMed Central

Chang, Yanli; Xu, Jianjun; Zhang, Qingyun

2017-01-01

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer. PMID:28943911

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

PubMed

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

2013-08-01

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals. Copyright © 2013 Elsevier Inc. All rights reserved.

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine.

PubMed

Saito, Yoshio; Kugenuma, Kenji; Tanaka, Makiko; Suzuki, Azusa; Saito, Isao

2012-06-01

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions

PubMed Central

TOISHI, Yuko; TSUNODA, Nobuo; NAGATA, Shun-ichi; KIRISAWA, Rikio; NAGAOKA, Kentaro; WATANABE, Gen; YANAGAWA, Yojiro; KATAGIRI, Seiji; TAYA, Kazuyoshi

2017-01-01

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs. PMID:29129877

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

PubMed

Lai, Wenqiang; Zeng, Qiao; Tang, Juan; Zhang, Maosheng; Tang, Dianping

2018-01-10

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B 1 (AFB 1 ). It is based on the just-in-time generation of an MnO 2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO 4 ) is converted into manganese dioxide (MnO 2 ) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO 4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO 2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO 4 . Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB 1 and magnetic beads carrying bovine serum albumin conjugated to AFB 1 are used for the determination of AFB 1 . In presence of AFB 1 , it will compete with the BSA-conjugated AFB 1 (on the magnetic beads) for the labeled antibody against AFB 1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB 1 concentrations in the range from 0.1 to 100 ng mL -1 , with a 0.1 ng mL -1 detection limit (at the 3S blank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

PubMed Central

Jackson, Colin R.; Tyler, Heather L.; Millar, Justin J.

2013-01-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample. PMID:24121617

Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.

PubMed

Jackson, Colin R; Tyler, Heather L; Millar, Justin J

2013-10-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Evaluation of reproductive function in Turkana women with enzyme immunoassays of urinary hormones in the field.

PubMed

Leslie, P W; Campbell, K L; Little, M A; Kigondu, C S

1996-02-01

The frequently reported observation that nomadic populations have lower fertility than their settled counterparts is often attributed to what are perceived as harsh, stressful conditions under which the nomads live. But the consequences of the hypothesized stresses for the reproductive biology or demography of these populations have been documented only a little. Traditionally, the Turkana of northwest Kenya are nomadic herders, but increasing numbers have settled on agricultural development schemes. We used an array of hormonal assays along with anthropometric indexes of nutritional status and interviews covering reproductive history, recent menstruation, diet, and health to compare reproductive function in nomadic and settled Turkana women. First morning urine samples were collected for three consecutive days during a series of surveys. Human choriogonadotropin (hCG; a marker for pregnancy), luteinizing hormone (LH; an indicator of ovulation), and pregnanediol glucuronide (PdG; an indicator of postovulatory luteal function) were assessed in the field with commercially available dipstick enzyme immunoassays. These assays along with the interview data allowed us to determine the reproductive status (e.g., pregnant or cycling, and if cycling, which phase of the ovarian cycle) of 166 nomadic and 194 settled Turkana women. The cross-sectional classifications allowed inferences of conception rates and normality of ovarian function. Follow-up surveys provided rates of pregnancy loss. Compared with the settled women, the nomadic women exhibited lower pregnancy rates and cycling nomadic women were less likely to show evidence of ovulation or luteal function. These results suggest that reproductive function of the nomadic women is diminished relative to the settled women. However, the settled women experienced a much higher rate of pregnancy loss, which may mean that their effective fecundability is in fact lower than that of the nomadic women. This study is the first to

Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine

PubMed Central

Barnes, Allan J.; Young, Sheena; Spinelli, Eliani; Martin, Thomas M.; Klette, Kevin L.; Huestis, Marilyn A.

2014-01-01

Introduction The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. Methods 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semiquantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 μg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. Results Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 μg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1 -10 μg/L. Performance at 5 μg/L was 92.2%, 98.1% and 97.4%, and for the 20 μg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 μg/L, and documented acceptable linearity from 5-25 μg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 μg/L); 4 showed moderate cross-reactivity (10–50% at 10 μg/L), 30 low cross-reactivity (<10% at 500 μg/L), and 27 <1% cross-reactivity at 500 μg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 μg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 μg/L) but below the manufacturer's recommended cutoff concentration (10 μg/L). Conclusion The Immunalysis HEIA K2 Spice kit

Simple immunoassay for detection of PCBs in transformer oil.

PubMed

Glass, Thomas R; Ohmura, Naoya; Taemi, Yukihiro; Joh, Takashi

2005-07-01

A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed.

Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.

PubMed

Stephen, Laurie; Schwarz, Emanuel; Guest, Paul C

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labour than in single immunoassays. This chapter details the methods to develop and manufacture a 5-plex immunoassay for the Luminex® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. An example will be given for the analysis of five hormones (glucagon-like peptide 1, growth hormone, insulin, leptin and thyroid-stimulating hormone) in serum samples from schizophrenia patients and controls.

Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus.

PubMed

Launay, David; Schmidt, Jean; Lepers, Sébastien; Mirault, Tristan; Lambert, Marc; Kyndt, Xavier; Reumaux, Dominique; Duhamel, Alain; Hachulla, Eric; Hatron, Pierre-Yves; Prin, Lionel; Dubucquoi, Sylvain

2010-07-04

Among anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE). Anti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases. Farr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE. Farr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment. Copyright 2010 Elsevier B.V. All rights reserved.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

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

PubMed

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

2015-11-01

The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12,481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/μL (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay.

PubMed

Tian, Jianniao; Zhou, Liujin; Zhao, Yanchun; Wang, Yuan; Peng, Yan; Zhao, Shulin

2012-04-15

A multicolor quantum dot (QD)-based nanosensor for multiplex detection of two tumor markers in a homogeneous format based on fluorescence polarization immunoassay was proposed. QDs520 and QDs620 were labeled alpha-fetoprotein(α-AFP) and carcinoembryonic antigen (CEA), respectively. After separated and purified by ultrafiltration, they were used in fluorescence polarization immunoassay for the simultaneous detection of human serum alpha-fetoprotein and carcinoembryonic antigen. Under the optimal conditions, the multi-analyte immunosensor had a wide linear range (from 0.5 ng mL(-1) to 500 ng mL(-1)) for both two tumor markers and good correlation (0.996 for α-AFP and 0.993 for CEA). The detection limits (LOD) were 0.36 ng mL(-1) for CEA and 0.28 ng mL(-1) for α-AFP (S/N=3). The carcinoembryonic antigen and fetoprotein in clinical serum samples were simultaneously detected. The results from 28 serum samples had a good agreement with enzyme-linked immunosorbent assay (ELISA). The relative standard deviation and the recovery suggested that the precision and the accuracy of this analytical method were satisfactory. This strategy with high sensitivity, good specificity, easy procedures and short analysis time shows great promise for clinical diagnoses and basic discovery. The application of QDs with longer fluorescence lifetime and small fluorescence polarization can be used for the determination of high molecular-weight substances which cannot be analyzed using dye fluorescence polarization immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers.

PubMed

Zhang, Bing; Liu, Bingqian; Liao, Jiayao; Chen, Guonan; Tang, Dianping

2013-10-01

A novel homogeneous immunoassay protocol was designed for quantitative monitoring of small molecular biotoxin (brevetoxin B, PbTx-2, as a model) by using target-responsive cargo release from polystyrene microsphere-gated mesoporous silica nanocontainer (MSN). Initially, monoclonal mouse anti-PbTx-2 capture antibody was covalently conjugated onto the surface of MSN (mAb-MSN), and the electroactive cargo (methylene blue, MB) was then trapped in the pores of mAb-MSN by using aminated polystyrene microspheres (APSM) based on the electrostatic interaction. Upon addition of target PbTx-2, the positively charged APSM was displaced from the negatively charged mAb-MSN because of the specific antigen-antibody reaction. Thereafter, the molecular gate was opened, and the trapped methylene blue was released from the pores. The released methylene blue could be monitored by using a square wave voltammetry (SWV) in a homemade microelectrochemical detection cell. Under optimal conditions, the SWV peak current increased with the increasing of PbTx-2 concentration in the range from 0.01 to 3.5 ng mL(-1) with a detection limit (LOD) of 6 pg mL(-1) PbTx-2 at the 3Sblank criterion. Intra- and interassay coefficients of variation with identical batches were ≤6% and 9.5%, respectively. The specificity and sample matrix interfering effects were acceptable. The analysis in 12 spiked seafood samples showed good accordance between results obtained by the developed immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method. Importantly, the target-responsive controlled release system-based electrochemical immunoassay (CRECIA) offers a promising scheme for the development of advanced homogeneous immunoassay without the sample separation and washing procedure.

Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker

PubMed Central

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

2006-01-01

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

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

PubMed

Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-06-07

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay.

PubMed

Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S; Hwang, Sung Hee; Holland, Erika B; Morisseau, Kevin; Pessah, Isaac N; Hammock, Bruce D; Gee, Shirley J

2016-04-05

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure.

Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

PubMed Central

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

2015-01-01

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

Sensitive, fast, and specific immunoassays for methyltestosterone detection.

PubMed

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

2015-04-29

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

Improved activity and thermo-stability of the horse radish peroxidase with graphene quantum dots and its application in fluorometric detection of hydrogen peroxide

NASA Astrophysics Data System (ADS)

Xiaoyan, Zhou; Yuanyuan, Jiang; Zaijun, Li; Zhiguo, Gu; Guangli, Wang

2016-08-01

Graphene quantum dots (GQDs) have received extensive concern in many fields such as optical probe, bioimaging and biosensor. However, few reports refer on the influence of GQDs on enzyme performance. The paper reports two kinds of graphene quantum dots (termed as GO-GQDs and N,S-GQDs) that were prepared by cutting of graphene oxide and pyrolysis of citric acid and L-cysteine, and their use for the horse radish peroxidase (HRP) modification. The study reveals that GO-GQDs and N,S-GQDs exhibit an opposite effect on the HRP performance. Only HRP modified with GO-GQDs offers an enhanced activity (more than 1.9 times of pristine enzyme) and thermo-stability. This is because GO-GQDs offer a larger conjugate rigid plane and fewer hydrophilic groups compared to N,S-GQDs. The characteristics can make GO-GQDs induce a proper conformational change in the HRP for the catalytic performance, improving the enzyme activity and thermo-stability. The HRP modified with green luminescent GO-GQDs was also employed as a biocatalyst for sensing of H2O2 by a fluorometric sensor. The colorless tetramethylbenzidine (TMB) is oxidized into blue oxidized TMB in the presence of H2O2 by the assistance of HRP/GO-GQDs, leading to an obvious fluorescence quenching. The fluorescence intensity linearly decreases with the increase of H2O2 concentration in the range from 2 × 10 - 9 to 2 × 10 - 4 M with the detection limit of 6.8 × 10 - 10 M. The analytical method provides the advantage of sensitivity, stability and accuracy compared with present H2O2 sensors based on the pristine HRP. It has been successfully applied in the determination of H2O2 in real water samples. The study also opens a new avenue for modification of enzyme activity and stability that offers great promise in applications such as biological catalysis, biosensing and enzyme engineering.

Streptavidin-functionalized capillary immune microreactor for highly efficient chemiluminescent immunoassay.

PubMed

Yang, Zhanjun; Zong, Chen; Ju, Huangxian; Yan, Feng

2011-11-07

A streptavidin functionalized capillary immune microreactor was designed for highly efficient flow-through chemiluminescent (CL) immunoassay. The functionalized capillary could be used as both a support for highly efficient immobilization of antibody and a flow cell for flow-through immunoassay. The functionalized inner wall and the capture process were characterized using scanning electron microscopy. Compared to conventional packed tube or thin-layer cell immunoreactor, the proposed microreactor showed remarkable properties such as lower cost, simpler fabrication, better practicality and wider dynamic range for fast CL immunoassay with good reproducibility and stability. Using α-fetoprotein as model analyte, the highly efficient CL flow-through immunoassay system showed a linear range of 3 orders of magnitude from 0.5 to 200 ng mL(-1) and a low detection limit of 0.1 ng mL(-1). The capillary immune microreactor could make up the shortcoming of conventional CL immunoreactors and provided a promising alternative for highly efficient flow-injection immunoassay. Copyright © 2011 Elsevier B.V. All rights reserved.

An organic-inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels.

PubMed

Su, Biling; Tang, Dianping; Tang, Juan; Li, Qunfang; Chen, Guonan

2011-10-01

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic-inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core-shell Fe(3)O(4)-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H(2)O(2) as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001-200 ng mL(-1) AFP with a low detection limit of 0.5 pg mL(-1) at 3s(B). Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL(-1) AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay. Copyright © 2011 Elsevier Inc. All rights reserved.

Mass spectrometric immunoassay

DOEpatents

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

2007-12-04

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

Mass spectrometric immunoassay

DOEpatents

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

2013-07-16

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

Mass spectrometric immunoassay

DOEpatents

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

2005-12-13

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jinwoo; Lee, Dohun; Oh, Eunkeu

2005-11-18

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

Fluorometric biosniffer (biochemical gas sensor) for breath acetone as a volatile indicator of lipid metabolism

NASA Astrophysics Data System (ADS)

Mitsubayashi, Kohji; Chien, Po-Jen; Ye, Ming; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro

2016-11-01

A fluorometric acetone biosniffer (biochemical gas sensor) for assessment of lipid metabolism utilizing reverse reaction of secondary alcohol dehydrogenase was constructed and evaluated. The biosniffer showed highly sensitivity and selectivity for continuous monitoring of gaseous acetone. The measurement of breath acetone concentration during fasting and aerobic exercise were also investigated. The acetone biosniffer provides a novel analytical tool for noninvasive evaluation of human lipid metabolism and it is also expected to use for the clinical and physiological applications such as monitoring the progression of diabetes.

[Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G].

PubMed

Sidorov, V L; Shvetsova, I V; Isakova, I V

2007-01-01

The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.

Extracellular enzyme activity in a willow sewage treatment system.

PubMed

Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

2012-12-01

This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

Use of a Fluorometric Imaging Plate Reader in high-throughput screening

NASA Astrophysics Data System (ADS)

Groebe, Duncan R.; Gopalakrishnan, Sujatha; Hahn, Holly; Warrior, Usha; Traphagen, Linda; Burns, David J.

1999-04-01

High-throughput screening (HTS) efforts at Abbott Laboratories have been greatly facilitated by the use of a Fluorometric Imaging Plate Reader. The FLIPR consists of an incubated cabinet with integrated 96-channel pipettor and fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorescence is imaged by a cooled CCD camera. The image data is downloaded from the camera and processed to average the signal form each well of the microtiter pate for each time point. The data is presented in real time on the computer screen, facilitating interpretation and trouble-shooting. In addition to fluorescence, the camera can also detect luminescence form firefly luciferase.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

PubMed

Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

2018-06-04

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

PubMed

Hirakawa, Koji; Katayama, Masaaki; Soh, Nobuaki; Nakano, Koji; Imato, Toshihiko

2006-01-01

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.

An organic thin film photodiode as a portable photodetector for the detection of alkylphenol polyethoxylates by a flow fluorescence-immunoassay on magnetic microbeads in a microchannel.

PubMed

Ishimatsu, Ryoichi; Naruse, Azusa; Liu, Rong; Nakano, Koji; Yahiro, Masayuki; Adachi, Chihaya; Imato, Toshihiko

2013-12-15

An organic thin film photodiode (OPD) was successfully employed as a portable photodetector in a competitive enzyme-linked immunosorbent assay (ELISA) of a class of nonionic surfactants, namely alkylphenol polyethoxylates (APnEOs) which are an environmental pollutant. Microbeads that were chemically immobilized with an anti-APnEOs antibody were used in the assay. The OPD consisted of a layer of copper phthalocyanine (CuPc), C60 and a second layer of bathocuproine (BCP) with a bulk heterojunction composed of CuPc and C60 prepared by a vapor deposition method on an indium-tin oxide coated glass substrate. The OPD showed an incident photon-current efficiency (IPCE) of approximately 19% for light at a wavelength of 585 nm. This relatively high IPCE at 585 nm makes it suitable for detecting the fluorescence of resorufin (λem=585 nm), the product of the competitive ELISA, produced through the enzymatic reaction of Amplex Red with horseradish peroxidase (HRP) and H2O2. A fluorometric detector was assembled on a microchip by combining the fabricated OPD and a commercial LED as a photodetector and a light source, respectively. The photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the concentration range from 0 to 8 μM. When the fabricated OPD was used as a portable photodetector, the competitive ELISA of APnEOs using HRP labeled APnEOs (HRP-APnEOs) was performed on magnetic microbeads on which surface an anti-APnEOs antibody had been immobilized. A typical sigmoidal calibration curve was obtained and the data were in good agreement with a numerical simulation, where the photocurrent of the OPD was plotted against the concentration of APnEOs, determined via the competitive ELISA. The detection limit of the immunoassay for APnEOs was approximately 2 and 4 ppb in batch and flow system, respectively. © 2013 Elsevier B.V. All rights reserved.

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay

PubMed Central

Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S.; Hwang, Sung Hee; Holland, Erika B.; Morisseau, Kevin; Pessah, Isaac N.; Hammock, Bruce D.; Gee, Shirley J.

2016-01-01

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4’-trichloro-2’-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4’-Cl and that replaced the 2’–OH with a –Cl were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library in order to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum #1155 and a heterologous competitive hapten where the 2’–OH group was substituted with a Cl. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21–6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (< 5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, TCS concentrations were measured in water samples following dilution. Biosolid samples were analyzed following dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure. PMID:26937944

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

PubMed

Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

2003-01-01

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

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

PubMed

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

2008-01-01

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

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

PubMed

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

2013-01-01

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

A fluorometric determination of urinary 17-hydroxycorticosteroids using benzamidine.

PubMed

Yamaguchi, Y; Seki, T

1984-10-01

A fluorometric determination of urinary 17-hydroxycorticosteroids using a reaction of benzamidine with compounds carrying the dihydroxyacetone side chain is described. The fluorescent compounds have excitation and emission maxima at 370 and 480 nm, respectively. The method includes enzymatic hydrolysis with beta-glucuronidase (EC 3.2.1.31, from Escherichia coli) and extraction with methylene chloride and generation of fluorescence in alkaline solution (pH 13.4). The specificity of the reaction was examined and the results were compared with those of an accepted method based on the Porter-Silber reaction (C. C. Porter and R. H. Silber, 1950, J. Biol. Chem. 185, 201-207). The coefficient of correlation was 0.945 with regression line of y = 0.91x + 0.7 mg/day (y, present method; x, Porter-Silber reaction method). Sensitivity of the reaction was 0.5 microgram/ml of standard or sample, mean recovery of cortisol added to five urine samples (5-micrograms addition) was 95%, and the coefficient of variation of the method (five repeated assays of sample with a value of 5.2 mg/liter) was 6.2%.

Fluorometric Index for Sensing Oil in the Sea Environment.

PubMed

Baszanowska, Emilia; Otremba, Zbigniew

2017-06-02

Excitation-emission matrix spectroscopy (EEMS) was applied to determine the fluorometric index (FI) as a parameter indicating the presence of a source of oil pollution in a specific area of the sea. Seawater from the Polish coast (the Baltic Sea) and the same water combined with various amounts of crude oil extracted from the Baltic Sea shelf ( Petrobaltic -type oil) were used in this study. The FI values were calculated for excitation and emission wavelengths found at the maximal peak, taking into account the natural seawater and the seawater artificially contaminated (for an oil-to-water ratio range of 0.5 × 10 -6 - 500 × 10 -6 ). The wavelength configurations (Ex/Em) (225/355 and 225/340) for the FI index were applied. It was found that, independent of the amount of oil, the FI achieves a higher value for natural seawater than for seawater that has had contact with oil. These results provide the basis to design a sensor signaling the appearance of oil in a defined sea area.

Regulation of Hydrolytic Enzyme Activity in Aquatic Microbial Communities Hosted by Carnivorous Pitcher Plants.

PubMed

Young, Erica B; Sielicki, Jessica; Grothjan, Jacob J

2018-04-20

Carnivorous pitcher plants Sarracenia purpurea host diverse eukaryotic and bacterial communities which aid in insect prey digestion, but little is known about the functional processes mediated by the microbial communities. This study aimed to connect pitcher community diversity with functional nutrient transformation processes, identifying bacterial taxa, and measuring regulation of hydrolytic enzyme activity in response to prey and alternative nutrient sources. Genetic analysis identified diverse bacterial taxa known to produce hydrolytic enzyme activities. Chitinase, protease, and phosphatase activities were measured using fluorometric assays. Enzyme activity in field pitchers was positively correlated with bacterial abundance, and activity was suppressed by antibiotics suggesting predominantly bacterial sources of chitinase and protease activity. Fungi, algae, and rotifers observed could also contribute enzyme activity, but fresh insect prey released minimal chitinase activity. Activity of chitinase and proteases was upregulated in response to insect additions, and phosphatase activity was suppressed by phosphate additions. Particulate organic P in prey was broken down, appearing as increasing dissolved organic and inorganic P pools within 14 days. Chitinase and protease were not significantly suppressed by availability of dissolved organic substrates, though organic C and N stimulated bacterial growth, resulting in elevated enzyme activity. This comprehensive field and experimental study show that pitcher plant microbial communities dynamically regulate hydrolytic enzyme activity, to digest prey nutrients to simpler forms, mediating biogeochemical nutrient transformations and release of nutrients for microbial and host plant uptake.

Morphological resonances for multicomponent immunoassays

NASA Astrophysics Data System (ADS)

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

1995-06-01

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

Validated low-volume aldosterone immunoassay tailored to GCLP-compliant investigations in small sample volumes.

PubMed

Schaefer, J; Burckhardt, B B; Tins, J; Bartel, A; Laeer, S

2017-12-01

Heart failure is well investigated in adults, but data in children is lacking. To overcome this shortage of reliable data, appropriate bioanalytical assays are required. Development and validation of a bioanalytical assay for the determination of aldosterone concentrations in small sample volumes applicable to clinical studies under Good Clinical Laboratory Practice. An immunoassay was developed based on a commercially available enzyme-linked immunosorbent assay and validated according to current bioanalytical guidelines of the EMA and FDA. The assay (range 31.3-1000 pg/mL [86.9-2775 pmol/L]) is characterized by a between-run accuracy from - 3.8% to - 0.8% and a between-run imprecision ranging from 4.9% to 8.9% (coefficient of variation). For within-run accuracy, the relative error was between - 11.1% and + 9.0%, while within-run imprecision ranged from 1.2% to 11.8% (CV). For parallelism and dilutional linearity, the relative error of back-calculated concentrations varied from - 14.1% to + 8.4% and from - 7.4% to + 10.5%, respectively. The immunoassay is compliant with the bioanalytical guidelines of the EMA and FDA and allows accurate and precise aldosterone determinations. As the assay can run low-volume samples, it is especially valuable for pediatric investigations.

Protein Multiplexed Immunoassay Analysis with R.

PubMed

Breen, Edmond J

2017-01-01

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex ® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex ® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex ® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays.

A Compact Immunoassay Platform Based on a Multicapillary Glass Plate

PubMed Central

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

2014-01-01

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

Evaluation of the C6 Lyme Enzyme Immunoassay for the Diagnosis of Lyme Disease in Children and Adolescents.

PubMed

Lipsett, Susan C; Branda, John A; McAdam, Alexander J; Vernacchio, Louis; Gordon, Caroline D; Gordon, Catherine R; Nigrovic, Lise E

2016-10-01

The commercially-available C6 Lyme enzyme immunoassay (EIA) has been approved to replace the standard whole-cell sonicate EIA as a first-tier test for the diagnosis of Lyme disease and has been suggested as a stand-alone diagnostic. However, the C6 EIA has not been extensively studied in pediatric patients undergoing evaluation for Lyme disease. We collected discarded serum samples from children and adolescents (aged ≤21 years) undergoing conventional 2-tiered testing for Lyme disease at a single hospital-based clinical laboratory located in an area endemic for Lyme disease. We performed a C6 EIA on all collected specimens, followed by a supplemental immunoblot if the C6 EIA result was positive but the whole-cell sonicate EIA result was negative. We defined a case of Lyme disease as either a clinician-diagnosed erythema migrans lesion or a positive standard 2-tiered serologic result in a patient with symptoms compatible with Lyme disease. We then compared the performance of the C6 EIA alone and as a first-tier test followed by immunoblot, with that of standard 2-tiered serology for the diagnosis of Lyme disease. Of the 944 specimens collected, 114 (12%) were from patients with Lyme disease. The C6 EIA alone had sensitivity similar to that of standard 2-tiered testing (79.8% vs 81.6% for standard 2-tiered testing; P = .71) with slightly lower specificity (94.2% vs 98.8% 2; P < .002). Addition of a supplemental immunoblot improved the specificity of the C6 EIA to 98.6%. For children and adolescents undergoing evaluation for Lyme disease, the C6 EIA could guide initial clinical decision making, although a supplemental immunoblot should still be performed. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

False Elevation of the Blood Tacrolimus Concentration, as Assessed by an Affinity Column-mediated Immunoassay (ACMIA), Led to Acute T Cell-mediated Rejection after Kidney Transplantation.

PubMed

Kono, Momoko; Hasegawa, Jumpei; Ogawa, Hina; Yoshikawa, Kanae; Ishiwatari, Ayumi; Wakai, Sachiko; Tanabe, Kazunari; Shirakawa, Hiroki

2018-05-01

Tacrolimus is the most commonly used immunosuppressant. Because of its narrow therapeutic range, it is necessary to frequently monitor its concentration. We report the case of a 25-year-old man who underwent kidney transplantation whose tacrolimus concentrations, as measured by an affinity column-mediated immunoassay, were falsely elevated. As we reduced the dose of tacrolimus, the recipient developed T cell-mediated rejection. Using the same blood samples, an enzyme-multiplied immunoassay technique showed that the patient's levels of tacrolimus were extremely low. A further examination indicated that the false increase in the tacrolimus concentration was likely due to an unknown interfering substance. We administered methylprednisolone and antithymocyte-globulin. The patient's serum creatinine level decreased and remained stable after these treatments.

The toxicology and immunology of detergent enzymes.

PubMed

Basketter, David; Berg, Ninna; Kruszewski, Francis H; Sarlo, Katherine; Concoby, Beth

2012-01-01

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

Microfluidic Analysis with Front-Face Fluorometric Detection for the Determination of Total Inorganic Iodine in Drinking Water.

PubMed

Inpota, Prawpan; Strzelak, Kamil; Koncki, Robert; Sripumkhai, Wisaroot; Jeamsaksiri, Wutthinan; Ratanawimarnwong, Nuanlaor; Wilairat, Prapin; Choengchan, Nathawut; Chantiwas, Rattikan; Nacapricha, Duangjai

2018-01-01

A microfluidic method with front-face fluorometric detection was developed for the determination of total inorganic iodine in drinking water. A polydimethylsiloxane (PDMS) microfluidic device was employed in conjunction with the Sandell-Kolthoff reaction, in which iodide catalyzed the redox reaction between Ce(IV) and As(III). Direct alignment of an optical fiber attached to a spectrofluorometer was used as a convenient detector for remote front-face fluorometric detection. Trace inorganic iodine (IO 3 - and I - ) present naturally in drinking water was measured by on-line conversion of iodate to iodide for determination of total inorganic iodine. On-line conversion efficiency of iodate to iodide using the microfluidic device was investigated. Excellent conversion efficiency of 93 - 103% (%RSD = 1.6 - 11%) was obtained. Inorganic iodine concentrations in drinking water samples were measured, and the results obtained were in good agreement with those obtained by an ICP-MS method. Spiked sample recoveries were in the range of 86%(±5) - 128%(±8) (n = 12). Interference of various anions and cations were investigated with tolerance limit concentrations ranging from 10 -6 to 2.5 M depending on the type of ions. The developed method is simple and convenient, and it is a green method for iodine analysis, as it greatly reduces the amount of toxic reagent consumed with reagent volumes in the microfluidic scale.

Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

USDA-ARS?s Scientific Manuscript database

A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

PubMed

Féraudet-Tarisse, Cécile; Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R; Dorner, Brigitte G; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

PubMed Central

Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R.; Dorner, Brigitte G.; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test. PMID:28700661

Effect of monensin on the levels of tachykinins and their processing enzyme activity in rat dorsal root ganglia.

PubMed

Chikuma, Toshiyuki; Inomata, Yuji; Tsuchida, Ken; Hojo, Hiroshi; Kato, Takeshi

2002-06-28

Th effect of monensin, which inhibits trans-Golgi function, on the levels of tachykinins and their processing enzyme activity was examined in organ-cultured rat dorsal root ganglia (DRG). Using an enzyme immunoassay method, we measured neurokinin A and substance P immunoreactivity in the DRG cultured for 72 h with and without 0.1 microM monensin. Both tachykinins were reduced in the DRG treated with monensin. Treatment with monensin also reduced the activity of carboxypeptidase E, which is one of the proteolytic processing enzymes of neuropeptides. These data suggest that proteolytic processing enzymes may in part modulate the biological activity of neuropeptides within a trans-Golgi apparatus.

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Dan; Wang, Jun; Wang, Limin

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic beads (MBs) immunosensing platform. The principle of this approach is based on the combination of MBs immuno-capture based enzyme activity assay and competitive immunoassay of total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target total BChE in a sample (mixture of OP-inhibited BChE and active BChE) competes with the BChE modified on themore » MBs to bind to the limited anti-BChE antibody labeled with quantum dots (QDs-anti-BChE), and followed by electrochemical stripping analysis of the bound QDs conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1~20 nM. Simultaneously, real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with microflow injection system after immuno-capture by MBs-anti-BChE conjugate. Therefore, the formed phosphorylated adduct (OP-BChE) can be estimated by the difference values of the total amount BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values, but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective and inexpensive quantification of phosphorylated adducts and enzyme inhibition for biomonitoring of OP and nerve agent exposures.« less

False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

PubMed

Arndt, Torsten; Grüner, Joachim; Schröfel, Stefanie; Stemmerich, Karsten

2012-11-30

Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented

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

PubMed

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

2013-01-01

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs. Copyright © 2012 Elsevier Inc. All rights reserved.

An Embedded Microretroreflector-Based Microfluidic Immunoassay Platform

PubMed Central

Raja, Balakrishnan; Pascente, Carmen; Knoop, Jennifer; Shakarisaz, David; Sherlock, Tim; Kemper, Steven; Kourentzi, Katerina; Renzi, Ronald F.; Hatch, Anson V.; Olano, Juan; Peng, Bi-Hung; Ruchhoeft, Paul; Willson, Richard

2017-01-01

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL. PMID:27025227

Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

PubMed Central

Frank, Ludmila A.

2010-01-01

Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects. PMID:22163526

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

PubMed

Clem, T R; Yolken, R H

1978-01-01

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

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates.

PubMed

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

2015-09-07

E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

Micromotor-based lab-on-chip immunoassays

NASA Astrophysics Data System (ADS)

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

2013-01-01

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

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

PubMed

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

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

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

PubMed Central

Clem, T R; Yolken, R H

1978-01-01

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories. Images PMID:342540

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

PubMed

Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

2016-12-01

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Human chorionic gonadotropin detection in cerebrospinal fluid of patients with a germinoma and its prognostic significance: assessment by using a highly sensitive enzyme immunoassay.

PubMed

Fukuoka, Kohei; Yanagisawa, Takaaki; Suzuki, Tomonari; Shirahata, Mitsuaki; Adachi, Jun-Ichi; Mishima, Kazuhiko; Fujimaki, Takamitsu; Katakami, Hideki; Matsutani, Masao; Nishikawa, Ryo

2016-11-01

OBJECTIVE Human chorionic gonadotropin (HCG) can be detected in a certain population of patients with a germinoma, but the frequency of germinoma HCG secretion and the prognostic value of HCG in the CSF are unknown. METHODS The authors measured HCG levels in sera and CSF in patients with a histologically confirmed germinoma by using a highly sensitive assay known as an immune complex transfer enzyme immunoassay (EIA), which is more than 100 times as sensitive as the conventional method, and they analyzed the correlation between HCG levels and the prognoses of patients with a germinoma. RESULTS HCG levels in sera and CSF of 35 patients with a germinoma were examined with the immune complex transfer EIA. The median CSF HCG levels in patients with a germinoma during the pretreatment and posttreatment evaluations were 192.5 pg/ml (range 1.2-13,116.5 pg/ml) and 18.7 pg/ml (1.2-283.9 pg/ml), respectively. Before treatment, the CSF HCG level was greater than the cutoff value in 85.7% of the patients with a germinoma. The authors compared survival rates among the patients by using a CSF HCG cutoff level of 1000 pg/ml, and the difference was statistically significant between the groups (p = 0.029, log-rank test). CONCLUSIONS Results of this study demonstrate that most germinomas secrete HCG. Patients with a germinoma that secretes higher amounts of HCG in their CSF experienced recurrence more frequently than those with lower CSF HCG levels.

Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children?

PubMed

Bae, Yoon Ju; Gaudl, Alexander; Jaeger, Sonia; Stadelmann, Stephanie; Hiemisch, Andreas; Kiess, Wieland; Willenberg, Anja; Schaab, Michael; von Klitzing, Kai; Thiery, Joachim; Ceglarek, Uta; Döhnert, Mirko; Kratzsch, Juergen

2016-05-01

Dysregulation of the adrenal cortex has been assessed with measurement of salivary cortisol. So far salivary cortisol is routinely measured with immunoassay (IA). However, liquid chromatography-tandem mass spectrometry (MS) is known to offer better specificity. We compared the concentrations of salivary cortisol measured by MS and IA at basal and stress induced conditions and evaluated reasons for the difference in method-dependent cortisol results. Saliva samples (n=2703) were collected from 169 children (age range: 8-14 years; 81 healthy children; 55 with internalizing and 33 with externalizing disorders) under circadian conditions and during the Trier Social Stress Test for Children (TSST-C). Biochemical analyses were performed with MS for cortisol and cortisone, IA (IBL, RE62011) for cortisol, and enzyme kinetic assay for α-amylase. MS and IA showed mostly comparable results for circadian activity and TSST-C response with similar statistical power. However, IA measured cortisol concentrations about 2.39-fold higher than MS. We found that this difference in measured values between MS and IA was mainly due to different standardization of IA compared to MS. In addition, at cortisol IA concentration below 5 nmol/L, cross-reactivity with cortisone was found to contribute to the lower concordance between MS and IA. Immunoassay and LC-MS/MS were largely comparable in the interpretation of salivary cortisol dynamics in stress research. But the IA method revealed a restricted accuracy in the measuring range below 5 nmol/L.

Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

PubMed

Stephen, Laurie; Guest, Paul C

2018-01-01

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

A magnetic/fluorometric bimodal sensor based on a carbon dots-MnO2 platform for glutathione detection

NASA Astrophysics Data System (ADS)

Xu, Yang; Chen, Xi; Chai, Ran; Xing, Chengfen; Li, Huanrong; Yin, Xue-Bo

2016-07-01

A novel magnetic/fluorometric bimodal sensor was built from carbon dots (CDs) and MnO2. The resulting sensor was sensitive to glutathione (GSH), leading to apparent enhancement of magnetic resonance (MR) and fluorescence signals along with visual changes. The bimodal detection strategy is based on the decomposition of the CDs-MnO2 through a redox reaction between GSH and MnO2. This process causes the transformation from non-MR-active MnO2 to MR-active Mn2+, and is accompanied by fluorescence restoration of CDs. Compared with a range of other CDs, the polyethylenimine (PEI) passivated CDs (denoted as pCDs) were suitable for detection due to their positive surface potential. Cross-validation between MR and fluorescence provided detailed information regarding the MnO2 reduction process, and revealed the three distinct stages of the redox process. Thus, the design of a CD-based sensor for the magnetic/fluorometric bimodal detection of GSH was emphasized for the first time. This platform showed a detection limit of 0.6 μM with a linear range of 1-200 μM in the fluorescence mode, while the MR mode exhibited a linear range of 5-200 μM and a GSH detection limit of 2.8 μM with a visible change being observed rapidly at 1 μM in the MR images. Furthermore, the introduction of the MR mode allowed the biothiols to be easily identified. The integration of CD fluorescence with an MR response was demonstrated to be promising for providing detailed information and discriminating power, and therefore extend the application of CDs in sensing and imaging.A novel magnetic/fluorometric bimodal sensor was built from carbon dots (CDs) and MnO2. The resulting sensor was sensitive to glutathione (GSH), leading to apparent enhancement of magnetic resonance (MR) and fluorescence signals along with visual changes. The bimodal detection strategy is based on the decomposition of the CDs-MnO2 through a redox reaction between GSH and MnO2. This process causes the transformation from non

Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

PubMed

Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els

2016-08-01

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential applications of immunoassays in studies of flatfish recruitment

NASA Astrophysics Data System (ADS)

Feller, Robert J.

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

Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

PubMed

Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

2017-01-01

Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

Electrokinetic Microstrirring to Enhance Immunoassays

NASA Astrophysics Data System (ADS)

Feldman, Hope; Sigurdson, Marin; Meinhart, Carl

2006-11-01

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

The Fundamental Flaws of Immunoassays and Potential Solutions Using Tandem Mass Spectrometry

PubMed Central

Hoofnagle, Andrew N.; Wener, Mark H.

2009-01-01

Immunoassays have made it possible to measure dozens of individual proteins and other analytes in human samples for help in establishing the diagnosis and prognosis of disease. In too many cases the results of those measurements are misleading and can lead to unnecessary treatment or missed opportunities for therapeutic interventions. These cases stem from problems inherent to immunoassays performed with human samples, which include a lack of concordance across platforms, autoantibodies, anti-reagent antibodies, and the high-dose hook effect. Tandem mass spectrometry may represent a detection method capable of alleviating many of the flaws inherent to immunoassays. We review our understanding of the problems associated with immunoassays on human specimens and describe methodologies using tandem mass spectrometry that could solve some of those problems. We also provide a critical discussion of the potential pitfalls of novel mass spectrometric approaches in the clinical laboratory. PMID:19538965

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

NASA Technical Reports Server (NTRS)

Ponce, Adrian

2003-01-01

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

Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

PubMed

Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

2018-05-23

Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

PubMed

Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

2017-10-01

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

Micromotor-based lab-on-chip immunoassays.

PubMed

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

2013-02-21

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

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

A fluorometric aptasensor for methamphetamine based on fluorescence resonance energy transfer using cobalt oxyhydroxide nanosheets and carbon dots.

PubMed

Saberi, Zeinab; Rezaei, Behzad; Faroukhpour, Hossein; Ensafi, Ali Ashghar

2018-05-17

Cobalt oxyhydroxide (CoOOH) nanosheets are efficient fluorescence quenchers due to their specific optical properties and high surface area. The combination of CoOOH nanosheets and carbon dots (CDs) has not been used in any aptasensor based on fluorescence quenching so far. An aptamer based fluorometric assay is introduced that is making use of fluorescent CDs conjugated to the aptamer against methamphetamine (MTA), and of CoOOH nanosheets which reduce the fluorescence of the CDs as a quencher. The results revealed that the conjugated CDs with aptamers were able to enclose the CoOOH nanosheets. Consequently, fluorescence is quenched. If the aptamer on the CD binds MTA, the CDs are detached from CoOOH nanosheets. As a result, fluorescence is restored proportionally to zhe MTA concentration. The fluorometric limit of detection is 1 nM with a dynamic range from 5 to 156 nM. The method was validated by comparing the results obtained by the new method to those obtained by ion mobility spectroscopy. Theoretical studies showed that the distance between CoOOH nanosheet and C-Ds is approximately 7.6 Å which can illustrate the possibility of FRET phenomenon. The interactions of MTA and the aptamer were investigated using molecular dynamic simulation (MDS). Graphical abstract Carbon dots (C-Ds) were prepared from grape leaves, conjugated to aptamer, and adsorbed on CoOOH nanosheets. So, the fluorescence of C-Ds is quenched. On addition of MTA, fluorescence is restored.

Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

NASA Astrophysics Data System (ADS)

Pandey, Binod Prasad

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL -1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl β-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1α)-D-Glc-1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A

78 FR 13347 - Clinical Chemistry and Clinical Toxicology Devices Panel of the Medical Devices Advisory...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

... as isoniazid test strips. Isoniazid test strips are considered pre-Amendment devices since they were... as methotrexate enzyme immunoassays. Methotrexate enzyme immunoassays are considered pre- Amendment... and PCP radioimmunoassays. PCP enzyme immunoassays and PCP radioimmunoassays are considered pre...

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

NASA Astrophysics Data System (ADS)

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

2008-10-01

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

Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX).

PubMed

Samara, Fatin; Gullett, Brian K; Harrison, Robert O; Chu, Andrew; Clark, George C

2009-04-01

Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo-p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (+/-0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (+/-0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (+/-34%), and 2,3,7-TriBDD at 84 (+/-36%).

Effect of Asian and Siberian ginseng on serum digoxin measurement by five digoxin immunoassays. Significant variation in digoxin-like immunoreactivity among commercial ginsengs.

PubMed

Dasgupta, Amitava; Wu, Sang; Actor, Jeffrey; Olsen, Margaret; Wells, Alice; Datta, Pradip

2003-02-01

Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48-0.68 ng/mL [0.6-0.9 nmol/L]; Siberian, 0.20-0.47 ng/mL [0.3-0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (e.g., for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.

State of the art of immunoassay methods for B-type natriuretic peptides: An update.

PubMed

Clerico, Aldo; Franzini, Maria; Masotti, Silvia; Prontera, Concetta; Passino, Claudio

2015-01-01

The aim of this review article is to give an update on the state of the art of the immunoassay methods for the measurement of B-type natriuretic peptide (BNP) and its related peptides. Using chromatographic procedures, several studies reported an increasing number of circulating peptides related to BNP in human plasma of patients with heart failure. These peptides may have reduced or even no biological activity. Furthermore, other studies have suggested that, using immunoassays that are considered specific for BNP, the precursor of the peptide hormone, proBNP, constitutes a major portion of the peptide measured in plasma of patients with heart failure. Because BNP immunoassay methods show large (up to 50%) systematic differences in values, the use of identical decision values for all immunoassay methods, as suggested by the most recent international guidelines, seems unreasonable. Since proBNP significantly cross-reacts with all commercial immunoassay methods considered specific for BNP, manufacturers should test and clearly declare the degree of cross-reactivity of glycosylated and non-glycosylated proBNP in their BNP immunoassay methods. Clinicians should take into account that there are large systematic differences between methods when they compare results from different laboratories that use different BNP immunoassays. On the other hand, clinical laboratories should take part in external quality assessment (EQA) programs to evaluate the bias of their method in comparison to other BNP methods. Finally, the authors believe that the development of more specific methods for the active peptide, BNP1-32, should reduce the systematic differences between methods and result in better harmonization of results.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-11-01

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

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

PubMed Central

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

2013-01-01

Background Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT). Methods We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose. Results In the validation study, mean intra-assay coefficients of variation (n = 162) were 15%, 13% and 10% for ‘low’ (6.78 µg/ml), ‘medium’ (18.18 µg/ml) and 'high’ (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n = 40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3–133%. Bloodspot adiponectin was stable for at least 30 months at −80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose. Conclusions Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood

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

PubMed Central

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

2012-01-01

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

Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an Enzyme-based Immunoassay

PubMed Central

Chowdhury, Basudev; Cho, Il-Hoon; Hahn, Noah; Irudayaraj, Joseph

2014-01-01

Background Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. Methods To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated Enzyme-based Immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. Results We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025 + 0.081, 0.023 + 0.006 and 0.001 + 0.0002 respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013 + 0.003 %) in comparison to healthy controls. Conclusion The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets. PMID:25441900

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9-12 Years Children.

PubMed

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9-11 years were determined by two methods of CPBA and HPLC. Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes.

Investigation of hydrazide derivatives inhibitory effect on peroxidase enzyme purified from turnip roots

NASA Astrophysics Data System (ADS)

Almaz, Züleyha; Öztekin, Aykut; Özdemir, Hasan

2017-04-01

Peroxidases (EC: 1.11.1.7) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group [1]. They are found in all cells and play a critical role in many biological processes, such as the host-defense mechanism [2]. Peroxidases (PODs) are widely used in clinical biochemistry, enzyme immunoassays, synthesis of various aromatic chemicals, treatment of waste water containing phenolic compounds [3, 4]. In this study, peroxidase enzyme was purified with Para amino benzohydrazide (PABH)-L-Tyrosine Sepharose 4B affinity chromatography to investigate the inhibitory effect of hydrazide derivatives on Turnip (Brassica rapa L.). IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzyme and inhibition type of these molecules were determined.

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

USDA-ARS?s Scientific Manuscript database

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

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...

Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction

PubMed Central

2014-01-01

Background Immunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids. Methods Cross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity. Results The Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement

Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Konstantinou, George N

2017-01-01

Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

NASA Astrophysics Data System (ADS)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-04-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

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

PubMed Central

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

2013-01-01

Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results. PMID:23114703

Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid.

PubMed

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

2013-01-01

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

Performance of the fourth-generation Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay for diagnosis of HIV infection in Southern Africa

PubMed Central

Piwowar-Manning, Estelle; Fogel, Jessica M.; Richardson, Paul; Wolf, Shauna; Clarke, William; Marzinke, Mark A.; Fiamma, Agnès; Donnell, Deborah; Kulich, Michal; Mbwambo, Jessie K.K.; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J.; Eshleman, Susan H.

2015-01-01

Background Fourth-generation HIV assays detect both antigen and antibody, facilitating detection of acute/early HIV infection. The Bio-Rad GS HIV Combo Ag/Ab assay (Bio-Rad Combo) is an enzyme immunoassay that simultaneously detects HIV p24 antigen and antibodies to HIV-1 and HIV-2 in serum or plasma. Objective To evaluate the performance of the Bio-Rad Combo assay for detection of HIV infection in adults from Southern Africa. Study design Samples were obtained from adults in Soweto and Vulindlela, South Africa and Dar es Salaam, Tanzania (300 HIV-positive samples; 300 HIV-negative samples; 12 samples from individuals previously classified as having acute/early HIV infection). The samples were tested with the Bio-Rad Combo assay. Additional testing was performed to characterize the 12 acute/early samples. Results All 300 HIV-positive samples were reactive using the Bio-Rad Combo assay; false positive test results were obtained for 10 (3.3%) of the HIV-negative samples (sensitivity: 100%, 95% confidence interval [CI]: 98.8–100%); specificity: 96.7%, 95% CI: 94.0–98.4%). The assay detected 10 of the 12 infections classified as acute/early. The two infections that were not detected had viral loads < 400 copies/mL; one of those samples contained antiretroviral drugs consistent with antiretroviral therapy. Conclusions The Bio-Rad Combo assay correctly classified the majority of study specimens. The specificity reported here may be higher than that seen in other settings, since HIV-negative samples were pre-screened using a different fourth-generation test. The assay also had high sensitivity for detection of acute/early infection. False-negative test results may be obtained in individuals who are virally suppressed. PMID:25542477

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

PubMed

Zella, D; Cavicchini, A; Cattaneo, E; Cimarelli, A; Bertazzoni, U

1995-02-01

The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

NASA Astrophysics Data System (ADS)

Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

2001-09-01

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

PubMed

Khalil, O S; Zurek, T F; Tryba, J; Hanna, C F; Hollar, R; Pepe, C; Genger, K; Brentz, C; Murphy, B; Abunimeh, N

1991-09-01

We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

A sensitive and quantitative element-tagged immunoassay with ICPMS detection.

PubMed

Baranov, Vladimir I; Quinn, Zoë; Bandura, Dmitry R; Tanner, Scott D

2002-04-01

We report a set of novel immunoassays in which proteins of interest can be detected using specific element-tagged antibodies. These immunoassays are directly coupled with an inductively coupled plasma mass spectrometer (ICPMS) to quantify the elemental (in this work, metal) component of the reacted tagged antibodies. It is demonstrated that these methods can detect levels of target proteins as low as 0.1-0.5 ng/mL and yield a linear response to protein concentration over 3 orders of magnitude.

An electrochemical immunoassay for the screening of celiac disease in saliva samples.

PubMed

Adornetto, Gianluca; Fabiani, Laura; Volpe, Giulia; De Stefano, Alessia; Martini, Sonia; Nenna, Raffaella; Lucantoni, Federica; Bonamico, Margherita; Tiberti, Claudio; Moscone, Danila

2015-09-01

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.

Clostridium difficile infection diagnostics - evaluation of the C. DIFF Quik Chek Complete assay, a rapid enzyme immunoassay for detection of toxigenic C. difficile in clinical stool samples.

PubMed

Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn

2016-11-01

Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician. © 2016 APMIS. Published by John Wiley & Sons Ltd.

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

PubMed

Piñeiro, Matilde; Pato, Raquel; Soler, Lourdes; Peña, Raquel; García, Natalia; Torrente, Carlos; Saco, Yolanda; Lampreave, Fermín; Bassols, Anna; Canalias, Francesca

2018-03-01

In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use. © 2018 American Society for Veterinary Clinical Pathology.

Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

NASA Astrophysics Data System (ADS)

Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

2016-04-01

Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to

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

EPA Science Inventory

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

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

PubMed Central

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

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

Electrochemical immunoassay for tumor markers based on hydrogels.

PubMed

Yin, Shuang; Ma, Zhanfang

2018-05-08

Hydrogel-based electrochemical immunoassays exhibit a large surface-to-volume ratio, excellent biocompatibility, unique stimuli-responsive behavior, high permeability and hydrophilicity and, thus, have shown great potential in the sensitive and accurate detection of tumor markers. Electrochemical immunosensing techniques for tumor markers based on hydrogels have greatly progressed in recent years. Areas covered: In this review, the authors describe the recent advances of hydrogel-based electrochemical immunosensing interface of tumor markers based on the different functions of hydrogels including conductive, catalytic, redox, stimuli-responsive and antifouling hydrogels. Expert commentary: Hydrogels have been successfully employed in electrochemical immunoassay of tumor markers, which is accountable to their unique properties. For further exploitation of hydrogel-based electrochemical biosensors, more variety of hydrogels need be fabricated with improved functionality.

BIOTIN INTERFERENCE WITH ROUTINE CLINICAL IMMUNOASSAYS: UNDERSTAND THE CAUSES AND MITIGATE THE RISKS.

PubMed

Samarasinghe, Shanika; Meah, Farah; Singh, Vinita; Basit, Arshi; Emanuele, Nicholas; Emanuele, Mary Ann; Mazhari, Alaleh; Holmes, Earle W

2017-08-01

The objectives of this report are to review the mechanisms of biotin interference with streptavidin/biotin-based immunoassays, identify automated immunoassay systems vulnerable to biotin interference, describe how to estimate and minimize the risk of biotin interference in vulnerable assays, and review the literature pertaining to biotin interference in endocrine function tests. The data in the manufacturer's "Instructions for Use" for each of the methods utilized by seven immunoassay system were evaluated. We also conducted a systematic search of PubMed/MEDLINE for articles containing terms associated with biotin interference. Available original reports and case series were reviewed. Abstracts from recent scientific meetings were also identified and reviewed. The recent, marked, increase in the use of over-the-counter, high-dose biotin supplements has been accompanied by a steady increase in the number of reports of analytical interference by exogenous biotin in the immunoassays used to evaluate endocrine function. Since immunoassay methods of similar design are also used for the diagnosis and management of anemia, malignancies, autoimmune and infectious diseases, cardiac damage, etc., biotin-related analytical interference is a problem that touches every area of internal medicine. It is important for healthcare personnel to become more aware of immunoassay methods that are vulnerable to biotin interference and to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases where a lab result does not correlate with the clinical scenario. FDA = U.S. Food & Drug Administration FT3 = free tri-iodothyronine FT4 = free thyroxine IFUs = instructions for use LH = luteinizing hormone PTH = parathyroid hormone SA/B = streptavidin/biotin TFT = thyroid function test TSH = thyroid-stimulating hormone.

Does the Alpha-defensin Immunoassay or the Lateral Flow Test Have Better Diagnostic Value for Periprosthetic Joint Infection? A Systematic Review.

PubMed

Eriksson, Hannah K; Nordström, Jakob; Gabrysch, Katja; Hailer, Nils P; Lazarinis, Stergios

2018-05-01

Measuring alpha-defensin concentrations in synovial fluid may help to diagnose periprosthetic joint infection (PJI). There are two commercially available methods for measuring alpha-defensin in synovial fluid: the enzyme-linked immunosorbent assay-based Synovasure® alpha-defensin immunoassay, which gives a numeric readout within 24 hours, and the Synovasure lateral flow test, which gives a binary readout within 20 minutes. There is no compilation of the existing literature to support the use of one of these two tests over the other. Does the immunoassay or the lateral flow test have better diagnostic value (sensitivity and specificity) in diagnosing PJI? We followed PRISMA guidelines and identified all studies on alpha-defensin concentration in synovial fluid as a PJI diagnostic marker, indexed to April 14, 2017, in PubMed, JSTOR, Google Scholar, and OVID databases. The search retrieved 1578 records. All prospective and retrospective studies on alpha-defensin as a PJI marker (PJI classified according to the criteria of the Musculoskeletal Infection Society) after THA or TKA were included in the analysis. All studies used only one of the two commercially available test methods, but none of them was comparative. After excluding studies with overlapping patient populations, four studies investigating the alpha-defensin immunoassay and three investigating the lateral flow test remained. Alpha-defensin immunoassay studies included 482 joints and lateral flow test studies included 119. The quality of the trials was assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. The heterogeneity among studies was evaluated by the I index, indicating that the heterogeneity of the included studies was low. Pooled sensitivity, specificity, positive and negative likelihood ratios, and receiver operating curves were calculated for each method and compared with each other. The alpha-defensin immunoassay had superior overall diagnostic value

Rapid immunoassays for diagnosis of heparin-induced thrombocytopenia: Comparison of diagnostic accuracy, reproducibility, and costs in clinical practice

PubMed Central

Bankova, Andriyana; Andres, Yvonne; Horn, Michael P.; Alberio, Lorenzo

2017-01-01

Background Immunoassays are crucial in the work-up of patients with suspected heparin-induced thrombocytopenia (HIT) and rapid tests have been recently developed. However, comparative data on diagnostic accuracy, reproducibility, and analytical costs of different immunoassays in clinical practice are limited. Methods Samples of 179 consecutive patients evaluated for suspected HIT in clinical practice using a polyspecific enzyme-linked immunoabsorbent assay (GTI diagnostics; ELISA) and a rapid particle gel immunoassay (PaGIA), were additionally analysed with a IgG-specific chemiluminescent immunoassay (AcuStar HIT-IgG). Presence of HIT was defined as a positive functional heparin-induced platelet aggregation test. Diagnostic accuracy was determined for low, intermediate and high thresholds as previously established (ELISA: optical density 0.4, 1.3, and 2.0 respectively; PaGIA: positive/negative, titre of 4, titre of 32; AcuStar HIT-IgG: 1.0 U/ml, 2.8, 9.4) and reproducibility was assessed by repeated measurements. Costs of test determination were calculated taking reagents, controls, and working time of technicians according to Swiss health care system into account. Results Data on PaGIA results were available for 171 patients (95.5%), ELISA for 144 patients (80.4%), and AcuStar HIT-IgG for 179 patients (100%). Sensitivity was above 95% for all assays at low and intermediate thresholds. Specificity increased with higher thresholds and was above 90% for all assays with intermediate and high thresholds. Specificity of AcuStar HIT-IgG (92.8%; 95% CI 87.7, 96.2) was significantly higher than PaGIA (83.0%; 95% CI 76.3, 88.5) and higher than ELISA (81.8%, 95% CI 74.2, 88.0) at low threshold (p<0.05). Reproducibility was adequate for all assays. Total costs per test were CHF 51.02 for ELISA, 117.70 for AcuStar HIT-IgG, and 83.13 for PaGIA. Conclusions We observed favourable diagnostic accuracy measures and a high reproducibility for PaGIA and AcuStar HIT

Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

PubMed

Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

2012-10-01

Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. Copyright © 2012 Elsevier Inc. All rights reserved.

Comparability of AMH levels among commercially available immunoassays

PubMed Central

Su, H. Irene; Sammel, Mary D.; Homer, Michael V.; Bui, Kim; Haunschild, Carolyn; Stanczyk, Frank Z.

2015-01-01

Objective To compare AMH levels among three commercially available AMH immunoassays (AMH Gen II, Beckman Coulter; Ultrasensitive AMH, AnshLab; picoAMH, AnshLab) Design Cross-sectional Setting Academic reproductive endocrinology program Patients 90 newly diagnosed breast cancer patients prior to cancer treatment Interventions None Outcome 1) proportion of detectable AMH levels by immunoassay, 2) comparability among assays Results At a mean age of 38.1, the median (interquartile range) for AMH levels for the cohort were 0.92 [1.35] ng/mL for the Gen II assay, 1.68 [2.30] ng/mL for the Ultrasensitive and 1.5 [2.41] ng/mL for the picoAMH assays. Significantly higher proportions of detectable AMH levels were observed with the picoAMH kit (97%) compared to both Gen II (84%) and Ultrasensitive (92%) assays. Although AMH results were highly correlated among assays (r=0.92–0.99), Gen II AMH levels were consistently lower than both Ultrasensitive and picoAMH levels. Moreover, as AMH levels increased, the magnitude of difference grew larger between Gen II and each of the other two assays. Conclusions Measurement of AMH levels with the picoAMH kit maximized detection at very low levels, particularly in contrast to the Gen II kit. Conversion of AMH levels from different immunoassays using regression equations is potentially highly inaccurate. PMID:24726216

Microarray-integrated optoelectrofluidic immunoassay system

PubMed Central

Han, Dongsik

2016-01-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection. PMID:27190571

Microarray-integrated optoelectrofluidic immunoassay system.

PubMed

Han, Dongsik; Park, Je-Kyun

2016-05-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

PubMed

Jacobson, R H; Downing, D R; Lynch, T J

1982-11-15

A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr; Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri; Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com

2016-04-18

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (Thismore » research was supported by Ataturk University. Project Number: BAP-2015/98).« less

Good performance of an immunoassay based method for nevirapine measurements in human breast milk.

PubMed

Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy; Pedersen, Court; Gerstoft, Jan; Katzenstein, Terese Lea

2011-07-01

Understanding the distribution of antiretro-virals in breastfeeding HIV-positive mothers is essential, both for prevention of mother-to-child HIV transmission and for research on the development of drug resistance. The ARK nevirapine (NVP)-test is an immunoassay method for nevirapine measurements, developed and validated for plasma use. In this study, the ARK NVP-test was evaluated for measurement of nevirapine concentrations in breast milk. High performance liquid chromatography (HPLC) is the method currently used to determine nevirapine in breast milk. This method, however, requires complicated extraction techniques. The ARK method employs an immunoassay technology and requires a small sample volume (40 μL) and no pre-treatment of the samples. Commercial enzyme and antibody were used and calibration standards and quality controls were prepared from pooled breast milk from HIV-uninfected women. Clinical samples from HIV-infected women receiving a single-dose of nevirapine were analyzed. Precision and accuracy were evaluated with two concentrations of quality control materials analyzed in three replicates on four different days and was <4%, and between 96.5% and 104.6%, respectively. Clinical samples were analyzed and CVs ranged from 0.0% to 11.1%. The median nevirapine concentration in breast milk 1 week post-partum was 0.29 μg/mL (range 0.11-0.90 μg/mL) in women treated with a single-dose of nevirapine. The ease of use and small sample volume makes the ARK assay an attractive alternative to HPLC analyses for determinations of nevirapine concentrations in breast milk.

An active lifestyle induces positive antioxidant enzyme modulation in peripheral blood mononuclear cells of overweight/obese postmenopausal women.

PubMed

Farinha, Juliano Boufleur; De Carvalho, Nélson Rodrigues; Steckling, Flávia Mariel; De Vargas, Liziane Da Silva; Courtes, Aline Alves; Stefanello, Sílvio Terra; Martins, Caroline Curry; Bresciani, Guilherme; Dos Santos, Daniela Lopes; Soares, Félix Alexandre Antunes

2015-01-15

The aim of this study was to investigate the effects of an active lifestyle on mitochondrial functioning, viability, bioenergetics, and redox status markers in peripheral blood mononuclear cells (PBMC) of overweight/ obese postmenopausal women. We performed a cross-sectional study with postmenopausal women aged 45–64 years and body mass index N 25 kg/m2, divided into physically active (n = 23) and sedentary (n = 12) groups. Mitochondria functioning and viability, bioenergetics and redox status parameters were assessed in PBMC with spectrophotometric and fluorometric assays. No differences were found in the enzyme activity of complexes I and II of the electron transport chain (ETC), mitochondrial superoxide dismutase (MnSOD) activity, methyl-tetrazolium reduction levels and reduced glutathione and oxidized glutathione levels between the groups. However, the physically active group presented higher levels of reactive oxygen species (ROS) (P= 0.04) and increased catalase (CAT) (P= 0.029), total (P= 0.011) and cytosolic SOD (CuZnSOD) (P= 0.009) activities. An active lifestyle that includes aerobic exercise for at least 30 min, three times per week may improve antioxidant enzyme activities in PBMC in overweight/obese postmenopausal women, without changes in the activity of the ETC enzymes. However, this low intensity physical activity is not able to induce relevant mitochondrial adaptations.

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies.

PubMed

Prince, H E; Ernst, C E; Hogrefe, W R

2000-01-01

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies. Copyright 2000 Wiley-Liss, Inc.

Evaluation of the Specificity of Two Enzyme Immunoassays for Coccidioidomycosis by Using Sera from a Region of Endemicity and a Region of Nonendemicity

PubMed Central

Ahn, YoonJi; McCotter, Orion; Gade, Lalitha; Hurst, Steven F.; Brandt, Mary E.; Park, Benjamin J.; Litvintseva, Anastasia P.

2015-01-01

Coccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico (n = 534), where CM is not endemic (who were not likely to have been exposed to Coccidioides spp.), compared to blood bank donors residing in Arizona (n = 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%; P < 0.001) but not IgM reactivity (3.4% versus 2.4%; P = 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly (P = 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures. PMID:26245352

Evaluation of a Newly Developed Lateral Flow Immunoassay for the Diagnosis of Cryptococcosis

PubMed Central

Lindsley, Mark D.; Mekha, Nanthawan; Baggett, Henry C.; Surinthong, Yupha; Autthateinchai, Rinrapas; Sawatwong, Pongpun; Harris, Julie R.; Park, Benjamin J.; Chiller, Tom; Poonwan, Natteewan

2011-01-01

Background. Cryptococcosis is a common opportunistic infection of human immunodeficiency virus (HIV)–infected individuals mostly occurring in resource-limited countries. This study compares the performance of a recently developed lateral flow immunoassay (LFA) to blood culture and enzyme immunoassay (EIA) for the diagnosis of cryptococcosis. Methods. Archived sera from 704 HIV-infected patients hospitalized for acute respiratory illness in Thailand were tested for cryptococcal antigenemia using EIA. All EIA-positive and a subset of EIA-negative sera were tested by LFA, with results recorded after 5 and 15 minutes incubation. Urine from patients with LFA- and EIA-positive sera was tested by LFA. Antigen results from patients with positive cryptococcal blood cultures were compared. Results. Of 704 sera, 92 (13%) were positive by EIA; among the 91 EIA-positive sera tested by LFA, 82 (90%) and 87 (96%) were LFA positive when read after 5 and 15 minutes, respectively. Kappa agreement of EIA and LFA for sera was 0.923 after 5 minutes and 0.959 after 15 minutes, respectively. Two of 373 EIA-negative sera were LFA positive at both time points. Of 74 urine specimens from EIA-positive patients, 52 (70.3%) were LFA positive. EIA was positive in 16 of 17 sera from blood culture–positive patients (94% sensitivity), and all sera were positive by LFA (100% sensitivity). Conclusions. A high level of agreement was shown between LFA and EIA testing of serum. The LFA is a rapid, easy-to-perform assay that does not require refrigeration, demonstrating its potential usefulness as a point-of-care assay for diagnosis of cryptococcosis in resource-limited countries. PMID:21810743

A fluorometric paper-based sensor array for the discrimination of heavy-metal ions.

PubMed

Feng, Liang; Li, Hui; Niu, Li-Ya; Guan, Ying-Shi; Duan, Chun-Feng; Guan, Ya-Feng; Tung, Chen-Ho; Yang, Qing-Zheng

2013-04-15

A fluorometric paper-based sensor array has been developed for the sensitive and convenient determination of seven heavy-metal ions at their wastewater discharge standard concentrations. Combining with nine cross-reactive BODIPY fluorescent indicators and array technologies-based pattern-recognition, we have obtained the discrimination capability of seven different heavy-metal ions at their wastewater discharge standard concentrations. After the immobilization of indicators and the enrichment of analytes, identification of the heavy-metal ions was readily acquired using a standard chemometric approach. Clear differentiation among heavy-metal ions as a function of concentration was also achieved, even down to 10(-7)M. A semi-quantitative estimation of the heavy-metal ion concentration was obtained by comparing color changes with a set of known concentrations. The sensor array was tentatively investigated in spiked tap water and sea water, and showed possible feasibility for real sample testing. Copyright © 2013 Elsevier B.V. All rights reserved.

Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.

PubMed

Nichkova, Mikaela; Galve, Roger; Marco, M-Pilar

2002-11-01

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay.

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

PubMed

de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

2017-01-18

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

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

PubMed

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

2014-09-01

A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 μIU/ml). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.

PubMed

Setlow, B; Korza, G; Setlow, P

2016-05-01

This study was to determine the sources, location and identity of α-glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. α-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl-α-d-glucoside (α-MUG) or α-MUG-6-phosphate hydrolysis fluorometrically. While α-MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with α-MUG showed that: (1) the α-glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α-glucosidase activity was only inside vegetative cells; (2) most α-glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti-α-glucosidase antiserum identified ≥2 α-glucosidases in spores and growing cells; (4) α-glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α-glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α-MUG, but glucose inhibited α-MUG uptake. α-MUG hydrolysis by G. stearothermophilus is by α-MUG uptake and hydrolysis by ≥2 α-glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance. © 2016 The Society for Applied Microbiology.

Separation of attogram terpenes by the capillary zone electrophoresis with fluorometric detection.

PubMed

Kubesová, Anna; Horká, Marie; Růžička, Filip; Slais, Karel; Glatz, Zdeněk

2010-11-12

An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium. Copyright © 2010 Elsevier B.V. All rights reserved.

Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

PubMed

Miyajima, Kumiko; Suzuki, Yurika; Miki, Daisuke; Arai, Moeka; Arakawa, Takahiro; Shimomura, Hiroji; Shiba, Kiyoko; Mitsubayashi, Kohji

2014-06-01

Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%. © 2013 Elsevier B.V. All rights reserved.

A quinoline-based fluorometric and colorimetric dual-modal pH probe and its application in bioimaging

NASA Astrophysics Data System (ADS)

Zhu, Qin; Li, Zhao; Mu, Lan; Zeng, Xi; Redshaw, Carl; Wei, Gang

2018-01-01

The compound (E)-8-hydroxyl-2-[(E)-2-(2, 4-dihydroxyphenyl)vinyl]-quinoline (1) has been developed as a fluorometric and colorimetric dual-modal probe for pH detection in solution and in vivo. Remarkable changes in the fluorescence intensity with large Stokes shifts and colorimetric responses were observed as a function of pH. The sensing mechanisms involving protonation and deprotonation processes over the acidic and alkaline pH ranges were confirmed by 1H NMR and IR spectroscopic analysis. Furthermore, the application of probe 1 for the imaging of live PC3 cells was successfully achieved. Test strips based on probe 1 were fabricated, and were found to act as a convenient and efficient pH test kits.

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

PubMed

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

2014-04-11

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 μg L(-1) to as low as 0.07 μg 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. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

NASA Astrophysics Data System (ADS)

Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

2000-03-01

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

Rapid and sensitive determination of beta-phenylethylamine in animal brains by high performance liquid chromatography with fluorometric detection.

PubMed

Taga, C; Tsuji, M; Nakajima, T

1989-05-01

A reversed phase HPLC method with fluorometric detection for the analysis of beta-phenylethylamine has been developed using p-methoxyphenylethylamine as an internal standard. Two columns, containing 200 microL of Dowex 50-X8 and Amberlite CG-50 respectively, were used to prepare a fraction containing beta-phenylethylamine. The recoveries of beta-phenylethylamine and p-methoxyphenylethylamine were 53.9 +/- 9.4% and 68.1 +/- 12.4%, respectively, and elution profile of p-methoxyphenylethylamine was sufficiently well correlated with that of beta-phenylethylamine. Regional distributions of beta-phenylethylamine in rat and mouse brains were determined. The highest concentrations were found in hypothalamus and hippocampus in both animals.

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

USGS Publications Warehouse

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

2005-01-01

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

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

USGS Publications Warehouse

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

2005-01-01

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

Use of Lambda Phage DNA as a Hybrid Internal Control in a PCR-Enzyme Immunoassay To Detect Chlamydia pneumoniae

PubMed Central

Pham, Dien G.; Madico, Guillermo E.; Quinn, Thomas C.; Enzler, Mark J.; Smith, Thomas F.; Gaydos, Charlotte A.

1998-01-01

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed Central

Ackerman, S B; Kelley, E A

1983-01-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units. Images PMID:6341399

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

Design and Fabrication of a PDMS Microchip Based Immunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Wanjun; Wang, Jun

2010-07-01

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

Comparison of pre-processing methods for multiplex bead-based immunoassays.

PubMed

Rausch, Tanja K; Schillert, Arne; Ziegler, Andreas; Lüking, Angelika; Zucht, Hans-Dieter; Schulz-Knappe, Peter

2016-08-11

High throughput protein expression studies can be performed using bead-based protein immunoassays, such as the Luminex® xMAP® technology. Technical variability is inherent to these experiments and may lead to systematic bias and reduced power. To reduce technical variability, data pre-processing is performed. However, no recommendations exist for the pre-processing of Luminex® xMAP® data. We compared 37 different data pre-processing combinations of transformation and normalization methods in 42 samples on 384 analytes obtained from a multiplex immunoassay based on the Luminex® xMAP® technology. We evaluated the performance of each pre-processing approach with 6 different performance criteria. Three performance criteria were plots. All plots were evaluated by 15 independent and blinded readers. Four different combinations of transformation and normalization methods performed well as pre-processing procedure for this bead-based protein immunoassay. The following combinations of transformation and normalization were suitable for pre-processing Luminex® xMAP® data in this study: weighted Box-Cox followed by quantile or robust spline normalization (rsn), asinh transformation followed by loess normalization and Box-Cox followed by rsn.

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

PubMed Central

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

Identification of Past and Recent Parvovirus B19 Infection in Immunocompetent Individuals by Quantitative PCR and Enzyme Immunoassays: a Dual-Laboratory Study

PubMed Central

Hedman, Lea; Dhanilall, Pravesh; Kantola, Kalle; Nurmi, Visa; Söderlund-Venermo, Maria; Brown, Kevin E.; Hedman, Klaus

2014-01-01

Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs. PMID:24403307

VLPs of HCV local isolates for HCV immunoassay diagnostic approach in Indonesia

NASA Astrophysics Data System (ADS)

Prasetyo, Afiono Agung

2017-01-01

Hepatitis C Virus (HCV) infection is a major global disease which often leads to morbidity and mortality. Low survival is related to the lack of adequate diagnostic because HCV infection is frequently asymptomatic and there are no specific diagnostic tests due to the fast transformation of the virus. Here, we investigated the VLPs (virus-like particles) of HCV local isolate as an immunoassay diagnostic approach to detect HCV infection, especially in Indonesia. The core, E1, and E2 of HCV local isolate genes were cloned and molecular analyzed, either as single or in recombinant-VLP form, to determine the molecular and chemical characteristics of each VLPs related to their potential use as an immunoassay detection method for HCV infection. The results indicated the molecular and chemical character of each VLPs are comparable. Conclusion: VLPs of HCV has the potential as an immunoassay diagnostic approach to detect HCV infection.

Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays

PubMed Central

2014-01-01

Background A challenge for drug of abuse testing is presented by ‘designer drugs’, compounds typically discovered by modifications of existing clinical drug classes such as amphetamines and cannabinoids. Drug of abuse screening immunoassays directed at amphetamine or methamphetamine only detect a small subset of designer amphetamine-like drugs, and those immunoassays designed for tetrahydrocannabinol metabolites generally do not cross-react with synthetic cannabinoids lacking the classic cannabinoid chemical backbone. This suggests complexity in understanding how to detect and identify whether a patient has taken a molecule of one class or another, impacting clinical care. Methods Cross-reactivity data from immunoassays specifically targeting designer amphetamine-like and synthetic cannabinoid drugs was collected from multiple published sources, and virtual chemical libraries for molecular similarity analysis were built. The virtual library for synthetic cannabinoid analysis contained a total of 169 structures, while the virtual library for amphetamine-type stimulants contained 288 compounds. Two-dimensional (2D) similarity for each test compound was compared to the target molecule of the immunoassay undergoing analysis. Results 2D similarity differentiated between cross-reactive and non-cross-reactive compounds for immunoassays targeting mephedrone/methcathinone, 3,4-methylenedioxypyrovalerone, benzylpiperazine, mephentermine, and synthetic cannabinoids. Conclusions In this study, we applied 2D molecular similarity analysis to the designer amphetamine-type stimulants and synthetic cannabinoids. Similarity calculations can be used to more efficiently decide which drugs and metabolites should be tested in cross-reactivity studies, as well as to design experiments and potentially predict antigens that would lead to immunoassays with cross reactivity for a broader array of designer drugs. PMID:24851137

How low can you go? Analytical performance of five automated testosterone immunoassays.

PubMed

La'ulu, Sonia L; Kalp, Kimberly J; Straseski, Joely A

2018-05-12

Testosterone is commonly measured using immunoassays, yet concerns with the accuracy and quality of testing by these methods exist, particularly for low testosterone concentrations. Study objectives were to evaluate selective performance characteristics, including functional sensitivity (FS), of 5 automated immunoassays for total testosterone. FS, imprecision, assay interference, limit of blank, linearity, and accuracy were assessed using the Abbott ARCHITECT i2000 SR , SIEMENS ADVIA Centaur and IMMULITE 2000, Beckman Coulter DxI 800, and Roche MODULAR E170. Comparisons to an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were performed using patient samples from men, women, boys, and girls. FS at 20% coefficient of variation (CV) for the ARCHITECT, Centaur, DxI, E170 and IMMULITE assays were 0.14, 1.23, 0.36, 0.77, 3.49 nmol/L, respectively. Total CVs for the 5-day imprecision study were ≤ 9.0% for all methods. All assays met manufacturer's claims for hemolysis, icterus, and lipemia interference and limit of blank. Dilution linearity studies had deviations from the target recoveries ranging from 3.4% (ARCHITECT) to 14.3% (DxI). Using National Institute of Standards and Technology Standard Reference Material 971, recoveries ranged from 79.2-149.2% (DxI, male and female, respectively). When compared to LC-MS/MS, more immunoassays under-recovered in men and women and over-recovered in boys and girls. Slopes ranged from 0.71 (IMMULITE, women) to 1.35 (DxI, boys). The combined average for percent bias was higher in boys (28.0%) than men (11.6%), women (22.8%), and girls (25.7%). Challenges with accurately measuring testosterone appear to remain for some immunoassays, but not all. While most immunoassays remain optimized for concentrations observed in healthy men, some showed acceptable performance when challenged at lower concentrations. Copyright © 2018. Published by Elsevier Inc.

Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

PubMed

Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

2016-06-01

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

PubMed Central

Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

2016-01-01

Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed. PMID:27525325

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

PubMed

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

2015-01-01

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

Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays. Applications, fundamentals, and optimization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driskell, Jeremy Daniel

2006-08-09

Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitivemore » immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical

Scheme for the selection of measurement uncertainty models in blood establishments' screening immunoassays.

PubMed

Pereira, Paulo; Westgard, James O; Encarnação, Pedro; Seghatchian, Jerard; de Sousa, Gracinda

2015-02-01

Blood establishments routinely perform screening immunoassays to assess safety of the blood components. As with any other screening test, results have an inherent uncertainty. In blood establishments the major concern is the chance of false negatives, due to its possible impact on patients' health. This article briefly reviews GUM and diagnostic accuracy models for screening immunoassays, recommending a scheme to support the screening laboratories' staffs on the selection of a model considering the intended use of the screening results (i.e., post-transfusion safety). The discussion is grounded on a "risk-based thinking", risk being considered from the blood donor selection to the screening immunoassays. A combination of GUM and diagnostic accuracy models to evaluate measurement uncertainty in blood establishments is recommended. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative Study of the New Colorimetric VITEK 2 Yeast Identification Card versus the Older Fluorometric Card and of CHROMagar Candida as a Source Medium with the New Card

PubMed Central

Aubertine, C. L.; Rivera, M.; Rohan, S. M.; Larone, D. H.

2006-01-01

The new VITEK 2 colorimetric card was compared to the previous fluorometric card for identification of yeast. API 20C was considered the “gold standard.” The new card consistently performed better than the older card. Isolates from CHROMagar Candida plates were identified equally as well as those from Sabouraud dextrose agar. PMID:16390976

Isotope labeled immunoassay for environmental chemical detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velez, M.M.

1994-05-06

Altrazine, one of the most heavily used agricultural pesticides in North America, has been identified as a major groundwater contaminant in the U.S. Research provides evidence that under certain conditions atrazine and some of its derivatives may prove to be carcinogenic and mutagenic. Immunossays are one of the most powerful of all analytical immunochemical techniques. They employ a wide range of methods to detect and quantitate antigens or antibodies, and to study the structure of antigens. With the appropriate assay, they can be remarkably quick and easy, to yield information that would be difficult to determine by other techniques. Themore » development of the appropriate assay; however, requires clean and precise separation of antigens bound to antibodies from those that remain free. Sensitive assays depend on quantification of these bound antigens at very low levels. We are making direct and competitive immunoassays with atrazine and its antibodies using accelerator mass spectrometry (AMS) in order to obtain a sensitive immunoassay for atrazine in environmental samples.« less

Comparative study of label-free electrochemical immunoassay on various gold nanostructures

NASA Astrophysics Data System (ADS)

Rafique, S.; Gao, C.; Li, C. M.; Bhatti, A. S.

2013-10-01

Electrochemical methods such as amperometry and impedance spectroscopy provide the feasibility of label-free immunoassay. However, the performance of electrochemical interfaces varies with the shape of gold nanostructures. In the present work three types of gold nanostructures including pyramid, spherical, and rod-like nanostructures were electrochemically synthesized on the gold electrode and were further transformed into immunosensor by covalent binding of antibodies. As a model protein, a cancer biomarker, Carcinoembryonic Antigen (CEA) was detected using amperometric and impedimetric techniques on three nanostructured electrodes, which enabled to evaluate and compare the immunoassay's performance. It was found that all three immunosensors showed improved linear electrochemical response to the concentration of CEA compared to bare Au electrode. Among all the spherical gold nanostructure based immunosensors displayed superior performance. Under optimal condition, the immunosensors exhibited a limit of detection of 4.1 pg ml-1 over a concentration range of five orders of magnitude. This paper emphasizes that fine control over the geometry of nanostructures is essentially important for high-performance electrochemical immunoassay.

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

PubMed

Ananworanich, Jintanat; Fletcher, James L K; Pinyakorn, Suteeraporn; van Griensven, Frits; Vandergeeten, Claire; Schuetz, Alexandra; Pankam, Tippawan; Trichavaroj, Rapee; Akapirat, Siriwat; Chomchey, Nitiya; Phanuphak, Praphan; Chomont, Nicolas; Michael, Nelson L; Kim, Jerome H; de Souza, Mark

2013-05-29

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

Multifunctional nanoparticles as simulants for a gravimetric immunoassay.

PubMed

Miller, Scott A; Hiatt, Leslie A; Keil, Robert G; Wright, David W; Cliffel, David E

2011-01-01

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au-tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.

Implementation of a rapid HIT immunoassay at a university hospital - Retrospective analysis of HIT laboratory orders in patients with thrombocytopenia.

PubMed

Black, Anne; Heimerl, Susanne; Oertli, Linnéa; Wilczek, Wolf; Greinacher, Andreas; Spannagl, Michael; Herr, Wolfgang; Hart, Christina

2017-10-01

Heparin-induced thrombocytopenia (HIT) is a rare cause of thrombocytopenia and a potentially life-threatening adverse drug reaction. Clinical overdiagnosis of HIT results in costly laboratory tests and anticoagulation. Criteria and algorithms for diagnosis are established, but their translation into clinical practice is still challenging. In a retrospective approach we studied all HIT related laboratory test requests within four years and evaluated data before (1st period, 24month) and after (2nd period, 24month) replacing particle gel immunoassay (PaGIA) and enzyme-linked immunosorbent assay (ELISA) by a chemiluminescent immunoassay (CLIA). HIT was confirmed by heparin-induced platelet activation (HIPA) test. Clinical pretest probability for HIT using an implemented simplified 4Ts score and platelet count were evaluated. Costs for laboratory tests and alternative anticoagulation were calculated. In 1850 patients with suspected HIT, 2327 laboratory orders were performed. In 87.2% of these orders an intermediate/high simplified 4Ts score was found. Thrombocytopenia was present in 87.1%. After replacing PaGIA and ELISA by CLIA the number of immunological and functional laboratory tests was reduced by 38.2%. The number of positive HIT immunoassays declined from 22.6% to 6.0%, while the number of positive HIPA tests among positive immunological tests increased by 19%. Altogether, acute HIT was confirmed in 59 patients. A decline in the use of alternative anticoagulants was observed in the 2nd period. Our study shows that in a university hospital setting HIT is well-known, but diagnosis requires a precise laboratory confirmation. Replacing PaGIA and ELISA by CLIA did not influence laboratory order behavior but results in reduced overall costs for laboratory diagnostics and alternative anticoagulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

PubMed

Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

2016-06-22

In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

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

PubMed Central

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

2014-01-01

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

Improvement of immunoassay detection system by using alternating current magnetic susceptibility

NASA Astrophysics Data System (ADS)

Kawabata, R.; Mizoguchi, T.; Kandori, A.

2016-03-01

A major goal with this research was to develop a low-cost and highly sensitive immunoassay detection system by using alternating current (AC) magnetic susceptibility. We fabricated an improved prototype of our previously developed immunoassay detection system and evaluated its performance. The prototype continuously moved sample containers by using a magnetically shielded brushless motor, which passes between two anisotropic magneto resistance (AMR) sensors. These sensors detected the magnetic signal in the direction where each sample container passed them. We used the differential signal obtained from each AMR sensor's output to improve the signal-to-noise ratio (SNR) of the magnetic signal measurement. Biotin-conjugated polymer beads with avidin-coated magnetic particles were prepared to examine the calibration curve, which represents the relation between AC magnetic susceptibility change and polymer-bead concentration. For the calibration curve measurement, we, respectively, measured the magnetic signal caused by the magnetic particles by using each AMR sensor installed near the upper or lower part in the lateral position of the passing sample containers. As a result, the SNR of the prototype was 4.5 times better than that of our previous system. Moreover, the data obtained from each AMR sensor installed near the upper part in the lateral position of the passing sample containers exhibited an accurate calibration curve that represented good correlation between AC magnetic susceptibility change and polymer-bead concentration. The conclusion drawn from these findings is that our improved immunoassay detection system will enable a low-cost and highly sensitive immunoassay.

Improvement of immunoassay detection system by using alternating current magnetic susceptibility.

PubMed

Kawabata, R; Mizoguchi, T; Kandori, A

2016-03-01

A major goal with this research was to develop a low-cost and highly sensitive immunoassay detection system by using alternating current (AC) magnetic susceptibility. We fabricated an improved prototype of our previously developed immunoassay detection system and evaluated its performance. The prototype continuously moved sample containers by using a magnetically shielded brushless motor, which passes between two anisotropic magneto resistance (AMR) sensors. These sensors detected the magnetic signal in the direction where each sample container passed them. We used the differential signal obtained from each AMR sensor's output to improve the signal-to-noise ratio (SNR) of the magnetic signal measurement. Biotin-conjugated polymer beads with avidin-coated magnetic particles were prepared to examine the calibration curve, which represents the relation between AC magnetic susceptibility change and polymer-bead concentration. For the calibration curve measurement, we, respectively, measured the magnetic signal caused by the magnetic particles by using each AMR sensor installed near the upper or lower part in the lateral position of the passing sample containers. As a result, the SNR of the prototype was 4.5 times better than that of our previous system. Moreover, the data obtained from each AMR sensor installed near the upper part in the lateral position of the passing sample containers exhibited an accurate calibration curve that represented good correlation between AC magnetic susceptibility change and polymer-bead concentration. The conclusion drawn from these findings is that our improved immunoassay detection system will enable a low-cost and highly sensitive immunoassay.

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

PubMed Central

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

2011-01-01

BACKGROUND Oral fluid (OF) is gaining prominence as an alternative matrix for monitoring drugs of abuse in the workplace, criminal justice, and driving under the influence of drugs programs. It is important to characterize assay performance and limitations of screening techniques for Δ9-tetrahydrocannabinol (THC) in OF. METHODS We collected OF specimens by use of the Quantisal™ OF collection device from 13 daily cannabis users after controlled oral cannabinoid administration. All specimens were tested with the Immunalysis Sweat/OF THC Direct ELISA and confirmed by 2-dimensional GC-MS. RESULTS The limit of detection was <1 µg/L THC equivalent, and the assay demonstrated linearity from 1 to 50 µg/L, with semiquantification to 200 µg/L. Intraplate imprecision (n = 7) ranged from 2.9% to 7.7% CV, and interplate imprecision (n = 20) was 3.0%–9.1%. Cross-reactivities at 4 µg/L were as follows: 11-hydroxy-THC, 198%; Δ8-tetrahydrocannabinol (Δ8-THC), 128%; 11-nor-9-carboxy-THC (THCCOOH), 121%; THC (target), 98%; cannabinol, 87%; THCCOOH-glucuronide, 11%; THC-glucuronide, 10%; and cannabidiol, 2.4%. Of 499 tested OF specimens, 52 confirmed positive (THC 2.0–290 µg/L), with 100% diagnostic sensitivity at the proposed Substance Abuse and Mental Health Services Administration screening cutoff of 4 µg/L cannabinoids and GC-MS cutoff of 2 µg/L THC. Forty-seven specimens screened positive but were not confirmed by 2D-GC-MS, yielding 89.5% diagnostic specificity and 90.6% diagnostic efficiency. Thirty-one of 47 unconfirmed immunoassay positive specimens were from 1 individual and contained >400 ng/L THCCOOH, potentially contributing to cross-reactivity. CONCLUSIONS The Immunalysis Sweat/OF THC Direct ELISA is an effective screening procedure for detecting cannabinoids in OF. PMID:20360126

An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

PubMed

Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

2013-05-01

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.

Hepatitis C prevalence and the significance of liver enzyme elevations in the insurance population.

PubMed

Stout, R L

1997-01-01

Liver enzyme elevation(s) are a common finding in the insurance applicant population. Hepatitis C infection results in histological and functional changes in the liver with both short and long term changes in serum liver enzyme levels. The prevalence of antibodies to HCV in the general population is estimated to be 4%. This paper reports on the prevalence of antibodies to HCV in the insurance applicant population and their relationship to the liver enzyme(s). Antibodies to HCV are present in 1.8% of a random sampling of insurance applicants. Alanine aminotransferase (ALT) elevations occur in 95.4% of all samples positive for antibodies to HCV. More than half of positive samples (56.7%) have ALT elevations of less than two time the upper range of normal. Antibody prevalence is lowest in samples with single enzyme elevation, 4.2%. In comparison, the prevalence is 16.4% in samples with all three enzymes, ALT, AST, and GGT, elevated. For maximal specificity two immunoassays, configured with different HCV antigens, should be performed sequentially on all positive applicant samples. HCV is the most prevalent, chronic viral infection in the insurance population. HCV prevalence is 40 times HIV prevalence. In an evaluation of enzyme reflex markers ALT was positive for antibodies to HCV 8.6% of the time while identifying 95.4% of HCV antibody positive applicants.

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...

Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.

PubMed

Sista, Ramakrishna S; Eckhardt, Allen E; Srinivasan, Vijay; Pollack, Michael G; Palanki, Srinivas; Pamula, Vamsee K

2008-12-01

A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels. Immunoassays on a digital microfluidic platform require the following basic operations: bead attraction, bead washing, bead retention, and bead resuspension. Several parameters such as magnetic field strength, pull force, position, and buffer composition were studied for effective bead operations. Dilution-based washing of magnetic beads was demonstrated by immobilizing the magnetic beads using a permanent magnet and splitting the excess supernatant using electrowetting. Almost 100% bead retention was achieved after 7776-fold dilution-based washing of the supernatant. Efficient resuspension of magnetic beads was achieved by transporting a droplet with magnetic beads across five electrodes on the platform and exploiting the flow patterns within the droplet to resuspend the beads. All the magnetic-bead droplet operations were integrated together to generate standard curves for sandwich heterogeneous immunoassays on human insulin and interleukin-6 (IL-6) with a total time to result of 7 min for each assay.

Heterogeneous Immunoassays Using Magnetic beads On a Digital Microfluidic Platform

PubMed Central

Sista, Ramakrishna S.; Eckhardt, Allen E.; Srinivasan, Vijay; Pollack, Michael G.; Palanki, Srinivas; Pamula, Vamsee K.

2009-01-01

A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels. Immunoassays on a digital microfluidic platform require the following basic operations: bead attraction, bead washing, bead retention, and bead resuspension. Several parameters such as magnetic field strength, pull force, position, and buffer composition were studied for effective bead operations. Dilution-based washing of magnetic beads was demonstrated by immobilizing the magnetic beads using a permanent magnet and splitting the excess supernatant using electrowetting. Almost 100% bead retention was achieved after 7776 fold dilution-based washing of the supernatant. Efficient resuspension of magnetic beads was achieved by transporting a droplet with magnetic beads across five electrodes on the platform and exploiting the flow patterns within the droplet to resuspend the beads. All the magnetic-bead droplet operations were integrated together to generate standard curves for sandwich heterogeneous immunoassays on Human Insulin and Interleukin-6 (IL-6) with a total time to result of seven minutes for each assay. PMID:19023486

Duplexed sandwich immunoassays on a fiber-optic microarray.

PubMed

Rissin, David M; Walt, David R

2006-03-30

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700 pM and 100 nM, while the working concentration range for lactoferrin was between 385 pM and 10 nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.

Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

PubMed Central

Obayashi, Yumiko; Wei Bong, Chui; Suzuki, Satoru

2017-01-01

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the

Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater.

PubMed

Obayashi, Yumiko; Wei Bong, Chui; Suzuki, Satoru

2017-01-01

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the

Magnetic luminescent nanoparticles as internal calibration for an immunoassay for ricin

NASA Astrophysics Data System (ADS)

Dosev, Dosi; Nichkova, Mikaela; Ma, Zhi-Ya; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2008-02-01

Fluorescence techniques rely on measurement of relative fluorescence units and require calibration to obtain reliable and comparable quantitative data. Fluorescent immunoassays are a very sensitive and convenient method of choice for rapid detection of biotoxins, such as ricin. Here we present the application of magnetic luminescent nanoparticles (MLNPs) with a magnetic core of Fe 3O 4 and a fluorescent shell of Eu:Gd IIO 3 as carriers for a nanobead-immunoassay for the detection of ricin with internal calibration. A sandwich immunoassay for ricin was performed on the surface of the MLNPs. The particles were functionalized with capture polyclonal antibodies. Anti-ricin antibodies labeled with Alexa Fluor dye were used as the detecting antibodies. After magnetic extraction, the amount of ricin bound to the particle surface was quantified and related to the fluorescence signal of the nanoparticles. In this new platform, the MLNPs have three main functions: (1) a probe for the specific extraction of the target analyte from the sample; (2) a carrier in the quantitative immunoassay with magnetic separation; and (3) an internal standard in the fluorescence measurement of the dye reporter. The MLNPs serve as an internal control for the total analysis including extraction and assay performance. This approach eliminates the experimental error inherent in particle extraction and measurement of absolute organic dye fluorescence intensities. All fluorescent measurements were performed in a microplate reader. The standard curve for ricin had a dynamic range from 20 ng/ml to 100 μg/ml with a detection limit of 5 ng/ml. The configuration that has been developed can be easily adapted to a high throughput miniaturized system.

Validated low-volume immunoassay for the reliable determination of direct renin especially valuable for pediatric investigations.

PubMed

Schaefer, J; Burckhardt, B B; Tins, J; Bartel, A; Laeer, S

2017-01-01

The pharmacotherapy of pediatric patients suffering from heart failure is extrapolated from adults due to missing data in children. Development and validation of a low-volume immunoassay for the reliable determination of renin. The immunoassay was validated according to international guidelines. The assay allows the reliable determination of renin in 40 μL plasma within a calibration range of 4-128 pg/mL. Between-run accuracy varied from -3.3 to +3.0% (relative error), while between-run precision ranged from 4.9 to 11.3% (coefficient of variation). The low-volume immunoassay facilitates the reliable collection of pharmacodynamic data in children.

Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen

PubMed Central

Sagar, Siddharth; Vishwanath, Shashidhar; Banerjee, Barnini; Eshwara, Vandana Kalwaje; Chawla, Kiran

2016-01-01

Introduction Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. Aim To compare the agreement between ELISA and CLIA in measurement of Anti–HBs antibody titers. Materials and Methods This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). Results Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. Conclusion Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in

Estimating False-Recent Classification for the Limiting-Antigen Avidity EIA and BED-Capture Enzyme Immunoassay in Vietnam: Implications for HIV-1 Incidence Estimates.

PubMed

Shah, Neha S; Duong, Yen T; Le, Linh-Vi; Tuan, Nguyen Anh; Parekh, Bharat S; Ha, Hoang Thi Thanh; Pham, Quang Duy; Cuc, Cao Thi Thu; Dobbs, Trudy; Tram, Tran Hong; Lien, Truong Thi Xuan; Wagar, Nick; Yang, Chunfu; Martin, Amy; Wolfe, Mitchell; Hien, Nguyen Tran; Kim, Andrea A

2017-06-01

Laboratory tests that can distinguish recent from long-term HIV infection are used to estimate HIV incidence in a population, but can potentially misclassify a proportion of long-term HIV infections as recent. Correct application of an assay requires determination of the proportion false recents (PFRs) as part of the assay characterization and for calculating HIV incidence in a local population using a HIV incidence assay. From April 2009 to December 2010, blood specimens were collected from HIV-infected individuals attending nine outpatient clinics (OPCs) in Vietnam (four from northern and five from southern Vietnam). Participants were living with HIV for ≥1 year and reported no antiretroviral (ARV) drug treatment. Basic demographic data and clinical information were collected. Specimens were tested with the BED capture enzyme immunoassay (BED-CEIA) and the Limiting-antigen (LAg)-Avidity EIA. PFR was estimated by dividing the number of specimens classified as recent by the total number of specimens; 95% confidence intervals (CI) were calculated. Specimens that tested recent had viral load testing performed. Among 1,813 specimens (north, n = 942 and south, n = 871), the LAg-Avidity EIA PFR was 1.7% (CI: 1.2-2.4) and differed by region [north 2.7% (CI: 1.8-3.9) versus south 0.7% (CI: 0.3-1.5); p = .002]. The BED-CEIA PFR was 2.3% (CI: 1.7-3.0) and varied by region [north 3.4% (CI: 2.4-4.7) versus south 1.0% (CI: 0.5-1.2), p < .001]. Excluding specimens with an undetectable VL, the LAg-Avidity EIA PFR was 1.2% (CI: 0.8-1.9) and the BED-CEIA PFR was 1.7% (CI: 1.2-2.4). The LAg-Avidity EIA PFR was lower than the BED-CEIA PFR. After excluding specimens with an undetectable VL, the PFR for both assays was similar. A low PFR should facilitate the implementation of the LAg-Avidity EIA for cross-sectional incidence estimates in Vietnam.

Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

PubMed

Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

1999-10-01

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

Estimating False-Recent Classification for the Limiting-Antigen Avidity EIA and BED-Capture Enzyme Immunoassay in Vietnam: Implications for HIV-1 Incidence Estimates

PubMed Central

Shah, Neha S.; Duong, Yen T.; Le, Linh-Vi; Tuan, Nguyen Anh; Parekh, Bharat S.; Thanh Ha, Hoang Thi; Pham, Quang Duy; Thu Cuc, Cao Thi; Dobbs, Trudy; Tram, Tran Hong; Xuan Lien, Truong Thi; Wagar, Nick; Yang, Chunfu; Martin, Amy; Wolfe, Mitchell; Hien, Nguyen Tran; Kim, Andrea A.

2017-01-01

Laboratory tests that can distinguish recent from long-term HIV infection are used to estimate HIV incidence in a population, but can potentially misclassify a proportion of long-term HIV infections as recent. Correct application of an assay requires determination of the proportion false recents (PFRs) as part of the assay characterization and for calculating HIV incidence in a local population using a HIV incidence assay. From April 2009 to December 2010, blood specimens were collected from HIV-infected individuals attending nine outpatient clinics (OPCs) in Vietnam (four from northern and five from southern Vietnam). Participants were living with HIV for ≥1 year and reported no antiretroviral (ARV) drug treatment. Basic demographic data and clinical information were collected. Specimens were tested with the BED capture enzyme immunoassay (BED-CEIA) and the Limiting-antigen (LAg)-Avidity EIA. PFR was estimated by dividing the number of specimens classified as recent by the total number of specimens; 95% confidence intervals (CI) were calculated. Specimens that tested recent had viral load testing performed. Among 1,813 specimens (north, n = 942 and south, n = 871), the LAg-Avidity EIA PFR was 1.7% (CI: 1.2–2.4) and differed by region [north 2.7% (CI: 1.8–3.9) versus south 0.7% (CI: 0.3–1.5); p = .002]. The BED-CEIA PFR was 2.3% (CI: 1.7–3.0) and varied by region [north 3.4% (CI: 2.4–4.7) versus south 1.0% (CI: 0.5–1.2), p < .001]. Excluding specimens with an undetectable VL, the LAg-Avidity EIA PFR was 1.2% (CI: 0.8–1.9) and the BED-CEIA PFR was 1.7% (CI: 1.2–2.4). The LAg-Avidity EIA PFR was lower than the BED-CEIA PFR. After excluding specimens with an undetectable VL, the PFR for both assays was similar. A low PFR should facilitate the implementation of the LAg-Avidity EIA for cross-sectional incidence estimates in Vietnam. PMID:28193090

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

PubMed Central

Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

1999-01-01

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of ≥0.650 and ≤0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

Fluorometric Measurement of Individual Stomata Activity and Transpiration via a “Brush-on”, Water-Responsive Polymer

PubMed Central

Seo, Minjeong; Park, Dong-Hoon; Lee, Chan Woo; Jaworski, Justyn; Kim, Jong-Man

2016-01-01

Much of atmospheric water originates from transpiration, the process by which plants release H2O from pores, known as stomata, that simultaneously intake CO2 for photosynthesis. Controlling stomatal aperture can regulate the extent of water transport in response to dynamic environmental factors including osmotic stress, temperature, light, and wind. While larger leaf regions are often examined, the extent of water vapor release from individual stomata remains unexplored. Using a “brush-on” sensing material, we can now assess transpiration using a water-responsive, polydiacetylene-based coating on the leaves surfaces. By eliciting a fluorometric signal to passing water vapor, we obtained information regarding the activity of individual stomata. In this demonstration, our results prove that this coating can identify the proportion of active stomata and the extent of transpirational diffusion of water in response to different conditions. PMID:27578430

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

PubMed

Yoo, Gu; Park, Min; Lee, Eun-Hang; Jose, Joachim; Pyun, Jae-Chul

2011-11-30

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

PubMed

Zhang, Hongyan; Zhang, Yan; Wang, Shuo

2008-08-20

Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

Fake news? Biotin interference in thyroid immunoassays.

PubMed

Koehler, Viktoria F; Mann, Ulrike; Nassour, Ayham; Alexander Mann, W

2018-05-29

We report on a 47 year old male patient with multiple sclerosis (MS) presenting in our outpatient neurology clinic in Frankfurt/Main for therapy evaluation. Before change of treatment laboratory investigations were performed. Thyroid function tests (TFTs) with a streptavidin/biotin based immunoassay revealed severe hyperthyroidism with positive thyroid autoantibodies suggestive for Graves' disease. Clinical presentation and thyroid sonography were unremarkable. Due to the discordance between clinical presentation and TFTs, we repeated medical history, in which the patient reported taking high-doses of biotin (300 mg/day) for MS. Recent studies with patients suffering from primary and secondary progressive MS, indicated promising effects of high-dose biotin on MS-related disability. In immunoassays relaying on streptavidin-biotin interaction, biotin intake can cause falsely high or low results. Two weeks after withdrawing biotin, biotin/streptavidin dependant assays showed no longer the biochemical picture of severe hyperthyroidism. Biotin intake should be paused for at least two to five days prior to the use of biotin/streptavidin dependant assays. Alternatively, non-biotin/streptavidin dependant assays (radioimmunoassay, gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry) may be used. Copyright © 2017. Published by Elsevier B.V.

Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

PubMed

Sachs, Ulrich J; von Hesberg, Jakob; Santoso, Sentot; Bein, Gregor; Bakchoul, Tamam

2011-12-01

Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

Investigation into low-level anti-rubella virus IgG results reported by commercial immunoassays.

PubMed

Dimech, Wayne; Arachchi, Nilukshi; Cai, Jingjing; Sahin, Terri; Wilson, Kim

2013-02-01

Since the 1980s, commercial anti-rubella virus IgG assays have been calibrated against a WHO International Standard and results have been reported in international units per milliliter (IU/ml). Laboratories testing routine patients' samples collected 100 samples that gave anti-rubella virus IgG results of 40 IU/ml or less from each of five different commercial immunoassays (CIA). The total of 500 quantitative results obtained from 100 samples from each CIA were compared with results obtained from an in-house enzyme immunoassay (IH-EIA) calibrated using the WHO standard. All 500 samples were screened using a hemagglutination inhibition assay (HAI). Any sample having an HAI titer of 1:8 or less was assigned a negative anti-rubella virus antibody status. If the HAI titer was greater than 1:8, the sample was tested in an immunoblot (IB) assay. If the IB result was negative, the sample was assigned a negative anti-rubella virus IgG status; otherwise, the sample was assigned a positive status. Concordance between the CIA qualitative results and the assigned negative status ranged from 50.0 to 93.8% and 74.5 to 97.8% for the assigned positive status. Using a receiver operating characteristic analysis with the cutoff set at 10 IU/ml, the estimated sensitivity and specificity ranged from 70.2 to 91.2% and 65.9 to 100%, respectively. There was poor correlation between the quantitative CIA results and those obtained by the IH-EIA, with the coefficient of determination (R(2)) ranging from 0.002 to 0.413. Although CIAs have been calibrated with the same international standard for more than 2 decades, the level of standardization continues to be poor. It may be time for the scientific community to reevaluate the relevance of quantification of anti-rubella virus IgG.

Investigation into Low-Level Anti-Rubella Virus IgG Results Reported by Commercial Immunoassays

PubMed Central

Arachchi, Nilukshi; Cai, Jingjing; Sahin, Terri; Wilson, Kim

2013-01-01

Since the 1980s, commercial anti-rubella virus IgG assays have been calibrated against a WHO International Standard and results have been reported in international units per milliliter (IU/ml). Laboratories testing routine patients' samples collected 100 samples that gave anti-rubella virus IgG results of 40 IU/ml or less from each of five different commercial immunoassays (CIA). The total of 500 quantitative results obtained from 100 samples from each CIA were compared with results obtained from an in-house enzyme immunoassay (IH-EIA) calibrated using the WHO standard. All 500 samples were screened using a hemagglutination inhibition assay (HAI). Any sample having an HAI titer of 1:8 or less was assigned a negative anti-rubella virus antibody status. If the HAI titer was greater than 1:8, the sample was tested in an immunoblot (IB) assay. If the IB result was negative, the sample was assigned a negative anti-rubella virus IgG status; otherwise, the sample was assigned a positive status. Concordance between the CIA qualitative results and the assigned negative status ranged from 50.0 to 93.8% and 74.5 to 97.8% for the assigned positive status. Using a receiver operating characteristic analysis with the cutoff set at 10 IU/ml, the estimated sensitivity and specificity ranged from 70.2 to 91.2% and 65.9 to 100%, respectively. There was poor correlation between the quantitative CIA results and those obtained by the IH-EIA, with the coefficient of determination (R2) ranging from 0.002 to 0.413. Although CIAs have been calibrated with the same international standard for more than 2 decades, the level of standardization continues to be poor. It may be time for the scientific community to reevaluate the relevance of quantification of anti-rubella virus IgG. PMID:23254301

25OHD analogues and vacuum blood collection tubes dramatically affect the accuracy of automated immunoassays

PubMed Central

Yu, Songlin; Cheng, Xinqi; Fang, Huiling; Zhang, Ruiping; Han, Jianhua; Qin, Xuzhen; Cheng, Qian; Su, Wei; Hou, Li’an; Xia, Liangyu; Qiu, Ling

2015-01-01

Variations in vitamin D quantification methods are large, and influences of vitamin D analogues and blood collection methods have not been systematically examined. We evaluated the effects of vitamin D analogues 25OHD2 and 3-epi 25OHD3 and blood collection methods on vitamin D measurement, using five immunoassay systems and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum samples (332) were selected from routine vitamin D assay requests, including samples with or without 25OHD2 or 3-epi 25OHD3, and analysed using various immunoassay systems. In samples with no 25OHD2 or 3-epi 25OHD3, all immunoassays correlated well with LC-MS/MS. However, the Siemens system produced a large positive mean bias of 12.5 ng/mL and a poor Kappa value when using tubes with clot activator and gel separator. When 25OHD2 or 3-epi 25OHD3 was present, correlations and clinical agreement decreased for all immunoassays. Serum 25OHD in VACUETTE tubes with gel and clot activator, as measured by the Siemens system, produced significantly higher values than did samples collected in VACUETTE tubes with no additives. Bias decreased and clinical agreement improved significantly when using tubes with no additives. In conclusion, most automated immunoassays showed acceptable correlation and agreement with LC-MS/MS; however, 25OHD analogues and blood collection tubes dramatically affected accuracy. PMID:26420221

25OHD analogues and vacuum blood collection tubes dramatically affect the accuracy of automated immunoassays.

PubMed

Yu, Songlin; Cheng, Xinqi; Fang, Huiling; Zhang, Ruiping; Han, Jianhua; Qin, Xuzhen; Cheng, Qian; Su, Wei; Hou, Li'an; Xia, Liangyu; Qiu, Ling

2015-09-30

Variations in vitamin D quantification methods are large, and influences of vitamin D analogues and blood collection methods have not been systematically examined. We evaluated the effects of vitamin D analogues 25OHD2 and 3-epi 25OHD3 and blood collection methods on vitamin D measurement, using five immunoassay systems and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum samples (332) were selected from routine vitamin D assay requests, including samples with or without 25OHD2 or 3-epi 25OHD3, and analysed using various immunoassay systems. In samples with no 25OHD2 or 3-epi 25OHD3, all immunoassays correlated well with LC-MS/MS. However, the Siemens system produced a large positive mean bias of 12.5 ng/mL and a poor Kappa value when using tubes with clot activator and gel separator. When 25OHD2 or 3-epi 25OHD3 was present, correlations and clinical agreement decreased for all immunoassays. Serum 25OHD in VACUETTE tubes with gel and clot activator, as measured by the Siemens system, produced significantly higher values than did samples collected in VACUETTE tubes with no additives. Bias decreased and clinical agreement improved significantly when using tubes with no additives. In conclusion, most automated immunoassays showed acceptable correlation and agreement with LC-MS/MS; however, 25OHD analogues and blood collection tubes dramatically affected accuracy.

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin

PubMed Central

Bartholomew, Rachel A.; Ozanich, Richard M.; Arce, Jennifer S.; Engelmann, Heather E.; Heredia-Langner, Alejandro; Hofstad, Beth A.; Hutchison, Janine R.; Jarman, Kristin; Melville, Angela M.; Victry, Kristin D.

2017-01-01

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 108 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 107 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use. PMID:28192054

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin.

PubMed

Bartholomew, Rachel A; Ozanich, Richard M; Arce, Jennifer S; Engelmann, Heather E; Heredia-Langner, Alejandro; Hofstad, Beth A; Hutchison, Janine R; Jarman, Kristin; Melville, Angela M; Victry, Kristin D; Bruckner-Lea, Cynthia J

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 10 8 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 10 7 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use.

A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay

PubMed Central

Tsai, Po-Yen; Lee, I-Chin; Hsu, Hsin-Yun; Huang, Hong-Yuan; Fan, Shih-Kang; Liu, Cheng-Hsien

2016-01-01

Here, we describe a technique to manipulate a low number of beads to achieve high washing efficiency with zero bead loss in the washing process of a digital microfluidic (DMF) immunoassay. Previously, two magnetic bead extraction methods were reported in the DMF platform: (1) single-side electrowetting method and (2) double-side electrowetting method. The first approach could provide high washing efficiency, but it required a large number of beads. The second approach could reduce the required number of beads, but it was inefficient where multiple washes were required. More importantly, bead loss during the washing process was unavoidable in both methods. Here, an improved double-side electrowetting method is proposed for bead extraction by utilizing a series of unequal electrodes. It is shown that, with proper electrode size ratio, only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. It allows using only about 25 magnetic beads in DMF immunoassay to increase the number of captured analytes on each bead effectively. In our human soluble tumor necrosis factor receptor I (sTNF-RI) model immunoassay, the experimental results show that, comparing to our previous results without using the proposed bead extraction technique, the immunoassay with low bead number significantly enhances the fluorescence signal to provide a better limit of detection (3.14 pg/ml) with smaller reagent volumes (200 nl) and shorter analysis time (<1 h). This improved bead extraction technique not only can be used in the DMF immunoassay but also has great potential to be used in any other bead-based DMF systems for different applications. PMID:26858807

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ross, G.S.; Elder, P.A.; McWha, J.A.

1987-09-01

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

Optimized Lateral Flow Immunoassay Reader for the Detection of Infectious Diseases in Developing Countries.

PubMed

Pilavaki, Evdokia; Demosthenous, Andreas

2017-11-20

Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassays can be used with great success for the detection of infectious diseases. However, for the quantification of their results an electronic reader is required. This paper presents an optimized handheld electronic reader for developing countries. It features a potentially low-cost, low-power, battery-operated device with no added optical accessories. The operation of this proof of concept device is based on measuring the reflected light from the lateral flow immunoassay and translating it into the concentration of the specific analyte of interest. Characterization of the surface of the lateral flow immunoassay has been performed in order to accurately model its response to the incident light. Ray trace simulations have been performed to optimize the system and achieve maximum sensitivity by placing all the components in optimum positions. A microcontroller enables all the signal processing to be performed on the device and a Bluetooth module allows transmission of the results wirelessly to a mobile phone app. Its performance has been validated using lateral flow immunoassays with influenza A nucleoprotein in the concentration range of 0.5 ng/mL to 200 ng/mL.

Interference of hawthorn on serum digoxin measurements by immunoassays and pharmacodynamic interaction with digoxin.

PubMed

Dasgupta, Amitava; Kidd, Laura; Poindexter, Brian J; Bick, Roger J

2010-08-01

Hawthorn is an herb indicated for treating cardiac illness. Because a patient taking digoxin may also take hawthorn, we investigated potential interference of hawthorn in serum digoxin measurements using immunoassays as well as pharmacodynamic interaction between hawthorn and digoxin. Hawthorn contains alkaloids that are structurally similar to digoxin and may interfere with serum digoxin measurement using immunoassays. In addition, hawthorn has cardioactive properties similar to digoxin. To study potential pharmacodynamic interaction between hawthorn and digoxin. The effects of hawthorn extract on serum digoxin measurements using Digoxin III (Abbott Laboratories, Abbott Park, Illinois) and the Tina-Quant digoxin assay (Roche Diagnostics, Indianapolis, Indiana) were investigated using 2 different brands of extract. To study the pharmacodynamic interaction between hawthorn and digoxin, we used an isolated adult rat cardiomyocyte system, measuring calcium transients by real-time fluorescence spectrophotometry. Hawthorn interfered only with the Digoxin III immunoassay but had no effect on the Tina-Quant assay. Both hawthorn extracts increased intracellular calcium levels, but the lack of additive response with digoxin suggests both may bind to the same site of Na, K adenosine triphosphatase. Because of interference of hawthorn with a digoxin immunoassay and pharmacodynamic interaction with digoxin, a patient receiving digoxin should avoid hawthorn.

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

PubMed Central

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

2008-01-01

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

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

PubMed

Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

2017-01-01

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

A novel pyridyl triphenylamine-BODIPY aldoxime: Naked-eye visible and fluorometric chemodosimeter for hypochlorite

NASA Astrophysics Data System (ADS)

Xu, Xiu-xiu; Qian, Ying

2017-08-01

An aldoxime containing fluorescent probe based on vinylpydine-appended triphenylamine-BODIPY has been designed and used for hypochlorite detection. OX-PPA-BODIPY was developed by introducing an aldoxime group into the 2-position of BODIPY, which can be used for the detection of hypochlorite with a sharp color change from pink to green. The attachment of 4-vinylpyridine moiety to triphenylamine-BODIPY constructs a fluorogen with desirable conjugated system. The probe, which displays extremely weak fluorescence owing to the Cdbnd N isomerization mechanism at 2-position of BODIPY, responds to HClO/ClO- through a dramatic enhancement of its fluorescence intensity. This new probe, a naked-eye visible and fluorometric chemodosimeter, exhibits high selectivity and sensitivity toward hypochlorite over other reactive oxygen species (ROS) and anions. The detection is accompanied by a 20-fold increase in fluorescent intensity (ΦF from 0.02 to 0.43). The detection limit of the probe for hypochlorite is 7.37 × 10- 7 M. Moreover, OX-PPA-BODIPY can be used to detect hypochlorite in real water samples.

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

Kajaysri, Jatuporn; Nokkaew, Weerapun

2014-03-01

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.

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

PubMed Central

Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

2006-01-01

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

Biomolecule-functionalized magnetic nanoparticles for flow-through quartz crystal microbalance immunoassay of aflatoxin B1.

PubMed

Wang, Li; Gan, Xian-Xue

2009-01-01

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B(1) antibody (anti-AFB(1))-functionalized magnetic core-shell Fe(3)O(4)/SiO(2) composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB(1), as a model protein, was initially covalently immobilized onto the Fe(3)O(4)/SiO(2) surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the AFB(1) concentration in the range of 0.3-7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.

A pilot study: subclinical hypothyroidism and free thyroid hormone measurement by immunoassay and mass spectrometry.

PubMed

Gounden, Verena; Jonklaas, Jacqueline; Soldin, Steven J

2014-03-20

The diagnosis of subclinical hypothyroidism is defined as the presence of an elevated thyroid stimulating hormone (TSH) with a normal free thyroxine (FT4) level. The commonly used direct analogue immunoassays for the measurement of FT4 have been shown to have poor performance at the upper and lower limits of the FT4 reference interval. The purpose of this pilot study was to investigate the percentage of individuals classified as having subclinical hypothyroidism with a standard immunoassay, that actually have low free thyroid hormone levels by mass spectrometry measurements. Outpatient samples with elevated TSH values and normal FT4 concentrations as per standard immunoassay methods were collected. FT4 and free triiodothyronine (FT3) analyses were performed on these samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty five percent (n=26) of patients (n=40) had (LC-MS/MS) FT4 or FT3 or both FT4 and FT3 values below mass spectrometry reference limits. Our findings indicate that the direct analogue immunoassay method for FT4 measurement results in a significant proportion of patients being misclassified as having subclinical hypothyroidism. Published by Elsevier B.V.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

PubMed

Zou, Fengming; Zhou, Hongjian; Tan, Tran Van; Kim, Jeonghyo; Koh, Kwangnak; Lee, Jaebeom

2015-06-10

A novel dual-mode immunoassay based on surface-enhanced Raman scattering (SERS) and fluorescence was designed using graphene quantum dot (GQD) labels to detect a tuberculosis (TB) antigen, CFP-10, via a newly developed sensing platform of linearly aligned magnetoplasmonic (MagPlas) nanoparticles (NPs). The GQDs were excellent bilabeling materials for simultaneous Raman scattering and photoluminescence (PL). The one-dimensional (1D) alignment of MagPlas NPs simplified the immunoassay process and enabled fast, enhanced signal transduction. With a sandwich-type immunoassay using dual-mode nanoprobes, both SERS signals and fluorescence images were recognized in a highly sensitive and selective manner with a detection limit of 0.0511 pg mL(-1).

Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.

PubMed

Yang, Xiaolan; Hu, Xiaolei; Xu, Bangtian; Wang, Xin; Qin, Jialin; He, Chenxiong; Xie, Yanling; Li, Yuanli; Liu, Lin; Liao, Fei

2014-06-17

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

PubMed

Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

2005-05-01

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3

Colorimetric stack pad immunoassay for bacterial identification.

PubMed

Eltzov, Evgeni; Marks, Robert S

2017-01-15

A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10 2 cellsmL -1 ). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

PubMed

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

2016-09-19

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems.

Does estradiol have an impact on the dipeptidyl peptidase IV enzyme activity of the Prevotella intermedia group bacteria?

PubMed

Fteita, Dareen; Könönen, Eija; Gürsoy, Mervi; Söderling, Eva; Gürsoy, Ulvi Kahraman

2015-12-01

Initiation and development of pregnancy-associated gingivitis is seemingly related to the microbial shift towards specific gram-negative anaerobes in subgingival biofilms. It is known that Prevotella intermedia sensu lato is able to use estradiol as an alternative source of growth instead of vitamin K. The aim of the present study was to investigate the impact of estradiol on the bacterial dipeptidyl peptidase IV (DPPIV) enzyme activity in vitro as a virulent factor of the Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca. In all experiments, 2 strains of each Prevotella species were used. Bacteria were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol and were allowed to build biofilms at an air-solid interface. DPPIV activities of biofilms were measured kinetically during 20 min using a fluorometric assay. The enzyme activity was later related to the amount of protein produced by the same biofilm, reflecting the biofilm mass. Estradiol significantly increased DPPIV activities of the 8 Prevotella strains in a strain- and dose-dependent manner. In conclusion, our in vitro experiments indicate that estradiol regulates the DPPIV enzyme activity of P. intermedia, P. nigrescens, P. pallens, and P. aurantiaca strains differently. Our results may, at least partly, explain the role of estradiol to elicit a virulent state which contributes to the pathogenesis of pregnancy-related gingivitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mobile Phone Ratiometric Imaging Enables Highly Sensitive Fluorescence Lateral Flow Immunoassays without External Optical Filters.

PubMed

Shah, Kamal G; Singh, Vidhi; Kauffman, Peter C; Abe, Koji; Yager, Paul

2018-05-14

Paper-based diagnostic tests based on the lateral flow immunoassay concept promise low-cost, point-of-care detection of infectious diseases, but such assays suffer from poor limits of detection. One factor that contributes to poor analytical performance is a reliance on low-contrast chromophoric optical labels such as gold nanoparticles. Previous attempts to improve the sensitivity of paper-based diagnostics include replacing chromophoric labels with enzymes, fluorophores, or phosphors at the expense of increased fluidic complexity or the need for device readers with costly optoelectronics. Several groups, including our own, have proposed mobile phones as suitable point-of-care readers due to their low cost, ease of use, and ubiquity. However, extant mobile phone fluorescence readers require costly optical filters and were typically validated with only one camera sensor module, which is inappropriate for potential point-of-care use. In response, we propose to couple low-cost ultraviolet light-emitting diodes with long Stokes-shift quantum dots to enable ratiometric mobile phone fluorescence measurements without optical filters. Ratiometric imaging with unmodified smartphone cameras improves the contrast and attenuates the impact of excitation intensity variability by 15×. Practical application was shown with a lateral flow immunoassay for influenza A with nucleoproteins spiked into simulated nasal matrix. Limits of detection of 1.5 and 2.6 fmol were attained on two mobile phones, which are comparable to a gel imager (1.9 fmol), 10× better than imaging gold nanoparticles on a scanner (18 fmol), and >2 orders of magnitude better than gold nanoparticle-labeled assays imaged with mobile phones. Use of the proposed filter-free mobile phone imaging scheme is a first step toward enabling a new generation of highly sensitive, point-of-care fluorescence assays.

Detection of cyclopiazonic acid (CPA) in maize by immunoassay

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (a-CPA) is a tremorgenic mycotoxin that is commonly produced by certain of the Aspergilli, in particular A. flavus, which is more widely known for production of the aflatoxins. Despite the fact that a-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for C...

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

PubMed

Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

2015-03-01

To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Gliadin Detection in Food by Immunoassay

NASA Astrophysics Data System (ADS)

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

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

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.

PubMed

Ingram, D T; Lamichhane, C M; Rollins, D M; Carr, L E; Mallinson, E T; Joseph, S W

1998-07-01

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 +/- 1 degree C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by

Development of a Colony Lift Immunoassay To Facilitate Rapid Detection and Quantification of Escherichia coli O157:H7 from Agar Plates and Filter Monitor Membranes

PubMed Central

Ingram, David T.; Lamichhane, Chinta M.; Rollins, David M.; Carr, Lewis E.; Mallinson, Edward T.; Joseph, Sam W.

1998-01-01

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3′,5,5′-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional

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...

Discordant Analytical Results Caused by Biotin Interference on Diagnostic Immunoassays in a Pediatric Hospital.

PubMed

Ali, Mahesheema; Rajapakshe, Deepthi; Cao, Liyun; Devaraj, Sridevi

2017-09-01

Recent studies have reported that biotin interferes with certain immunoassays. In this study, we evaluated the analytical interference of biotin on immunoassays that use streptavidin-biotin in our pediatric hospital. We tested the effect of different concentrations of biotin (1.5-200 ng/ml) on TSH, Prolactin, Ferritin, CK-MB, β-hCG, Troponin I, LH, FSH, Cortisol, Anti-HAV antibody (IgG and IgM), assays on Ortho Clinical Diagnostic Vitros 5600 Analyzer. Biotin (up to 200 ng/mL) did not significantly affect Troponin I and HAV assays. Biotin (up to 12.5 ng/ml) resulted in <10% bias in CK-MB, β-hCG, AFP, Cortisol, Ferritin assays and biotin >6.25 ng/mL significantly affected TSH (>20% bias) assay. Prolactin was significantly affected even at low levels (Biotin 1.5 ng/mL). Thus, we recommend educating physicians about biotin interference in common immunoassays and adding an electronic disclaimer. © 2017 by the Association of Clinical Scientists, Inc.

Enhancement of Fluorescence-Based Sandwich Immunoassay Using Multilayered Microplates Modified with Plasma-Polymerized Films

PubMed Central

Yano, Kazuyoshi; Iwasaki, Akira

2016-01-01

A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2) was performed with the multilayered microplates, resulting in specific and 88-fold–enhanced fluorescence detection. PMID:28029144

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

PubMed

Kim, Myoung-Ho; Choi, Suk-Jung

2015-04-15

In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps. Copyright © 2014 Elsevier B.V. All rights reserved.

Microsphere-Based Immunoassay for the Detection of Azaspiracids

PubMed Central

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

2014-01-01

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

Galactomannan Enzymatic Immunoassay Cross-Reactivity Caused by Prototheca Species

PubMed Central

Van den Bossche, D.; Hendrickx, M.; De Becker, A.; Jacobs, R.; Naessens, A.; Piérard, D.

2012-01-01

We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis. PMID:22837317

Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

PubMed

Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

2015-01-01

The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

[Detecting the markers of HIV infection with the new enzyme immunoassay diagnostic kit "DS-EIA-HIV-AB-AG-SPECTRUM" at the laboratories of AIDS prevention and control centers in the Volga Federal District].

PubMed

Ivanova, N I; Peksheva, O Iu

2009-03-01

A possibility of simultaneously detecting specific antibodies to HIV-1 and HIV-2 by enzyme immunoassay (EIA) at lower concentrations than those by immunoblotting (IB), and well as an additional possibility of earlier diagnosis of HIV infection, by identifying the HIV-1 antigen p24 lay the foundation of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system made by OOO "Research-and-Production Association "Diagnosticheskiye Sistemy" (Diagnostic Systems). These peculiarities were compared with those of IB at a number of laboratories of AIDS prevention and control centers in the Volga Federal District, by using native serum/plasma samples and a specially designed control panel. The analysis of the conducted studies to identify HIV-1 and HIV-2 antibodies and HIV-1 antigen p24 in 65 plasma/serum samples in the "DS-EIA-HIV-AB-AG-SPECTRUM" and "LIA-HIV-1/2" (OOO "Niarmedik plus") test systems while confirming the positive result indicated agreement in 57 (87.7%) cases. The diagnostic possibilities of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system versus the "New Lav-Blot I" one to make a laboratory diagnosis of HIV infection were studied. Irrefragable answers as to the availability of HIV-1 markers in the study serum samples on the enciphered panel were provided by IB in 73.3% of cases and EIA in 92%.

IFSA: a microfluidic chip-platform for frit-based immunoassay protocols

NASA Astrophysics Data System (ADS)

Hlawatsch, Nadine; Bangert, Michael; Miethe, Peter; Becker, Holger; Gärtner, Claudia

2013-03-01

Point-of-care diagnostics (POC) is one of the key application fields for lab-on-a-chip devices. While in recent years much of the work has concentrated on integrating complex molecular diagnostic assays onto a microfluidic device, there is a need to also put comparatively simple immunoassay-type protocols on a microfluidic platform. In this paper, we present the development of a microfluidic cartridge using an immunofiltration approach. In this method, the sandwich immunoassay takes place in a porous frit on which the antibodies have immobilized. The device is designed to be able to handle three samples in parallel and up to four analytical targets per sample. In order to meet the critical cost targets for the diagnostic market, the microfluidic chip has been designed and manufactured using high-volume manufacturing technologies in mind. Validation experiments show comparable sensitivities in comparison with conventional immunofiltration kits.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

PubMed

Wheaton, Catharine J; Mylniczenko, Natalie D; Rimoldi, John M; Gadepalli, Rama S V S; Hart, R; O'Hara, Bobbi R; Evans, Andrew N

2018-01-31

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays

Comparison of sensor structures for the signal amplification of surface plasmon resonance immunoassay using enzyme precipitation

NASA Astrophysics Data System (ADS)

Yang, Chih-Tsung; Thierry, Benjamin

2015-12-01

Surface plasmon resonance (SPR) biosensing has been successfully applied for the label-free detection of a broad range of bioanalytes ranging from bacteria, cell, exosome, protein and nucleic acids. When it comes to the detection of small molecules or analytes found at low concentration, amplification schemes are desirable to enhance binding signals and in turn increase sensitivity. A number of SPR signal amplification schemes have been developed and validated; however, little effort has been devoted to understanding the effect of the SPR sensor structures on the amplification of binding signals and therefore on the overall sensing performance. The physical phenomenon of long-range SPR (LRSPR) relies on the propagation of coupled surface plasmonic waves on the opposite sides of a nanoscale-thick noble metal film suspended between two dielectrics with similar refractive indices. Importantly, as compared with commonly used conventional SPR (cSPR), LRSPR is not only characterized by a longer penetration depth of the plasmonic waves in the analyzed medium but also by a greater sensitivity to bulk refractive index changes. In this work, an immunoassay signal amplification platform based on horseradish peroxidase (HRP) catalyzed precipitation was utilized to investigate the sensing performance with regards to cSPR and LRSPR. The enzymatic precipitation of 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 was used to amplify SPR signals. The structure-function relationship of cSPR and LRSPR sensors is presented for both standard refractometric measurements and the enzymatic precipitation scheme. Experimental data shows that despite its inherent higher sensitivity to bulk refractive index changes and higher figure of merit, LRSPR was characterized by a lower angular sensitivity in the model enzymatic amplification scheme used here.

Magneto-actuated immunoassay for the detection of Mycobacterium fortuitum in hemodialysis water.

PubMed

Brugnera, Michelle Fernanda; Bundalian, Reynaldo; Laube, Tamara; Julián, Esther; Luquin, Marina; Zanoni, Maria Valnice Boldrin; Pividori, Maria Isabel

2016-06-01

This paper addresses a sensitive method for the detection of mycobacteria in hemodialysis water samples based on a magneto-actuated immunoassay with optical readout. In this approach, micro (2.8μm) sized magnetic particles were modified with an antibody against the lipoarabinomannan (LAM) located in the mycobacterial cell wall. The system relies on the immunocapturing of the mycobacteria with the tailored antiLAM magnetic particles to pre-concentrate the bacteria from the hemodialysis samples throughout an immunological reaction. The performance of the immunomagnetic separation on the magnetic carrier was evaluated using confocal microscopy to study the binding pattern, as well as a magneto-actuated immunoassay with optical readout for the rapid detection of the bacteria in spiked hemodialysis samples. In this approach, the antiLAM polyclonal antibody was labeled with fluorescein isothiocyanate. The optical readout was achieved by the incubation with a secondary anti-fluorescein antibody labeled with peroxidase as optical reporter. The magneto-actuated immunoassay was able to detect mycobacteria contamination in hemodialysis water at a limit of detection of 13CFUmL(-1) in a total assay time of 3h without any previous culturing pre-enrichment step. Copyright © 2016 Elsevier B.V. All rights reserved.

Colorimetric and fluorometric determination of uric acid based on the use of nitrogen-doped carbon quantum dots and silver triangular nanoprisms.

PubMed

Wang, Yanying; Yang, Yan; Liu, Wei; Ding, Fang; Zhao, Qingbiao; Zou, Ping; Wang, Xianxiang; Rao, Hanbing

2018-05-04

A dual-read detection system is described for non-enzymatic and non-aggregation based analysis of uric acid (UA). Silver triangular nanoprisms (AgTNPs) were used as colorimetric probes, while the reduction in the fluorescence of nitrogen-doped carbon quantum dots (N-CQDs) served as the fluorometric readout. The absorption band of the AgTNPs overlaps the emission band of N-CQDs (with a peak at 440 nm). Therefore, fluorescence is reduced owing to an inner filter effect. The AgTNPs are etched if exposed to H 2 O 2 , and round nanodiscs are formed. In the presence of UA, etching of the AgTNPs is suppressed because the facets of the AgTNPs are coated with UA. The absorbance, best measured at 683 nm, increases with the concentration of the pre-added UA. The colorimetric assay works in the 0.1-45 μM UA concentration range, and the fluorometric assay between 1 and 42 μM of UA. The respective detection limits are 50 and 200 nM, respectively. The probe can be used for direct visualization of UA. The method was successfully applied to the determination of UA in urine samples. Graphical abstract The fluorescence of nitrogen-doped carbon quantum dots (N-CQDs) is quenched by AgTNPs (silver triangular nanoprisms). As the AgTNPs are etched by H 2 O 2 , fluorescence recovers in the system after H 2 O 2 is added, and also undergoes a color change. Uric acid (UA) protects the AgTNPs from etching because the facets of the AgTNPs are coated with UA. The fluorescence of N-CQDs decreases. Thus, a dual-read probe is developed for determination of UA.

Immobilization of microorganisms for detection by solid-phase immunoassays.

PubMed Central

Ibrahim, G F; Lyons, M J; Walker, R A; Fleet, G H

1985-01-01

Several cultures of gram-negative and gram-positive bacteria were successfully immobilized with titanous hydroxide. The immobilization efficiency for the microorganisms investigated in saline and broth media ranged from 80.2 to 99.9%. The immobilization of salmonellae was effective over a wide pH range. The presence of buffers, particularly phosphate buffer, drastically reduced the immobilization rate. However, buffers may be added to immunoassay systems after immobilization of microorganisms. The immobilization process involved only one step, i.e., shaking 100 microliter of culture with 50 microliter of titanous hydroxide suspension in polystyrene tubes for only 10 min. The immobilized cells were so tenaciously bound that vigorous agitation for 24 h did not result in cell dissociation. The nonspecific binding of 125I-labeled antibody from rabbits and 125I-labeled protein A by titanous hydroxide was inhibited in the presence of 2% gelatin and amounted to only 5.6 and 3.9%, respectively. We conclude that this immobilization procedure is a potentially powerful tool which could be utilized in solid-phase immunoassays concerned with the diagnosis of microorganisms. PMID:3900128

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Impact of nucleic acid testing relative to antigen/antibody combination immunoassay on the detection of acute HIV infection.

PubMed

De Souza, Mark S; Phanuphak, Nittaya; Pinyakorn, Suteeraporn; Trichavaroj, Rapee; Pattanachaiwit, Supanit; Chomchey, Nitiya; Fletcher, James L; Kroon, Eugene D; Michael, Nelson L; Phanuphak, Praphan; Kim, Jerome H; Ananworanich, Jintanat

2015-04-24

To assess the addition of HIV nucleic acid testing (NAT) to fourth-generation (4thG) HIV antigen/antibody combination immunoassay in improving detection of acute HIV infection (AHI). Participants attending a major voluntary counseling and testing site in Thailand were screened for AHI using 4thG HIV antigen/antibody immunoassay and sequential less sensitive HIV antibody immunoassay. Samples nonreactive by 4thG antigen/antibody immunoassay were further screened using pooled NAT to identify additional AHI. HIV infection status was verified following enrollment into an AHI study with follow-up visits and additional diagnostic tests. Among 74 334 clients screened for HIV infection, HIV prevalence was 10.9% and the overall incidence of AHI (N = 112) was 2.2 per 100 person-years. The inclusion of pooled NAT in the testing algorithm increased the number of acutely infected patients detected, from 81 to 112 (38%), relative to 4thG HIV antigen/antibody immunoassay. Follow-up testing within 5 days of screening marginally improved the 4thG immunoassay detection rate (26%). The median CD4 T-cell count at the enrollment visit was 353 cells/μl and HIV plasma viral load was 598 289 copies/ml. The incorporation of pooled NAT into the HIV testing algorithm in high-risk populations may be beneficial in the long term. The addition of pooled NAT testing resulted in an increase in screening costs of 22% to identify AHI: from $8.33 per screened patient to $10.16. Risk factors of the testing population should be considered prior to NAT implementation given the additional testing complexity and costs.

Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

PubMed

Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

2017-04-15

Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel pyridyl triphenylamine-BODIPY aldoxime: Naked-eye visible and fluorometric chemodosimeter for hypochlorite.

PubMed

Xu, Xiu-Xiu; Qian, Ying

2017-08-05

An aldoxime containing fluorescent probe based on vinylpydine-appended triphenylamine-BODIPY has been designed and used for hypochlorite detection. OX-PPA-BODIPY was developed by introducing an aldoxime group into the 2-position of BODIPY, which can be used for the detection of hypochlorite with a sharp color change from pink to green. The attachment of 4-vinylpyridine moiety to triphenylamine-BODIPY constructs a fluorogen with desirable conjugated system. The probe, which displays extremely weak fluorescence owing to the CN isomerization mechanism at 2-position of BODIPY, responds to HClO/ClO - through a dramatic enhancement of its fluorescence intensity. This new probe, a naked-eye visible and fluorometric chemodosimeter, exhibits high selectivity and sensitivity toward hypochlorite over other reactive oxygen species (ROS) and anions. The detection is accompanied by a 20-fold increase in fluorescent intensity (Φ F from 0.02 to 0.43). The detection limit of the probe for hypochlorite is 7.37×10 -7 M. Moreover, OX-PPA-BODIPY can be used to detect hypochlorite in real water samples. Copyright © 2017 Elsevier B.V. All rights reserved.

False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

PubMed

Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

2017-05-01

Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Jun; Li, Zhaohui

2011-09-20

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstratemore » the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 ul sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.« less

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

PubMed Central

Niederkofler, Eric E.; Phillips, David A.; Krastins, Bryan; Kulasingam, Vathany; Kiernan, Urban A.; Tubbs, Kemmons A.; Peterman, Scott M.; Prakash, Amol; Diamandis, Eleftherios P.; Lopez, Mary F.; Nedelkov, Dobrin

2013-01-01

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories. PMID:24278387

Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia.

PubMed

Leroux, Dorothée; Hezard, Nathalie; Lebreton, Aurélien; Bauters, Anne; Suchon, Pierre; de Maistre, Emmanuel; Biron, Christine; Huisse, Marie-Genevieve; Ternisien, Catherine; Voisin, Sophie; Gruel, Yves; Pouplard, Claire

2014-09-01

A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT. © 2014 John Wiley & Sons Ltd.

Nano-immunoassay with improved performance for detection of cancer biomarkers

DOE PAGES

Krasnoslobodtsev, Alexey V.; Torres, Maria P.; Kaur, Sukhwinder; ...

2015-01-01

Nano-immunoassay utilizing surface-enhanced Raman scattering (SERS) effect is a promising analytical technique for the early detection of cancer. In its current standing the assay is capable of discriminating samples of healthy individuals from samples of pancreatic cancer patients. Further improvements in sensitivity and reproducibility will extend practical applications of the SERS-based detection platforms to wider range of problems. In this report, we discuss several strategies designed to improve performance of the SERS-based detection system. We demonstrate that reproducibility of the platform is enhanced by using atomically smooth mica surface as a template for preparation of capture surface in SERS sandwichmore » immunoassay. Furthermore, the assay's stability and sensitivity can be further improved by using either polymer or graphene monolayer as a thin protective layer applied on top of the assay addresses. The protective layer renders the signal to be more stable against photo-induced damage and carbonaceous contamination.« less

Chitosan-coated polystyrene microplate for covalent immobilization of enzyme.

PubMed

Zhang, Yaodong; Li, Li; Yu, Caihong; Hei, Tingting

2011-10-01

Microplates made of polystyrene have been widely used for immunoassays. Protein molecules that have been immobilized on a hydrophobic polystyrene microplate by passive adsorption lose their activity and suffer considerable denaturation. A new chitosan-coated microplate suitable for the covalent immobilization of enzymes has been developed. The primary amino groups of chitosan were exploited for this covalent coupling of proteins. The optical transmittance of the chitosan-coated microplate, at wavelengths of 400-800 nm, was estimated to be suitable for its application in chromogenic reaction-based bioassays. The immobilization efficiency of the chitosan-coated microplate was demonstrated to be far superior to that of a conventional microplate when tested using acetylcholinesterase (AChE) and β-glucosidase as model biomolecules, and the chitosan-coated microplate may thus have potential applications in biosensing and bioreactor systems. © Springer-Verlag 2011

Comparability of antimüllerian hormone levels among commercially available immunoassays.

PubMed

Su, H Irene; Sammel, Mary D; Homer, Michael V; Bui, Kim; Haunschild, Carolyn; Stanczyk, Frank Z

2014-06-01

To compare antimüllerian hormone (AMH) levels among three commercially available AMH immunoassays: AMH Gen II (Beckman Coulter), Ultrasensitive AMH (Ansh Labs), and picoAMH (Ansh Labs). Cross-sectional. Academic reproductive endocrinology program. 90 newly diagnosed breast cancer patients before cancer treatment. None. Proportion of detectable AMH levels by immunoassay, and comparability among assays. At a mean age of 38.1 years, the median (interquartile range) AMH level for the cohort was 0.92 [1.35] ng/mL for the Gen II assay, 1.68 [2.30] ng/mL for the Ultrasensitive assay, and 1.52 [2.41] ng/mL for the picoAMH assay. Significantly higher proportions of detectable AMH levels were observed with the picoAMH kit (97%) compared with both the Gen II (84%) and Ultrasensitive (92%) assays. Although the AMH results were highly correlated among the assays (r = 0.92-0.99), the Gen II AMH levels were consistently lower than both Ultrasensitive and picoAMH levels. Moreover, as AMH levels increased, the magnitude of difference grew larger between Gen II and each of the other two assays. Measurement of AMH levels with the picoAMH kit maximized detection at very low levels, particularly in contrast with the Gen II kit. Conversion of AMH levels from different immunoassays using regression equations is potentially highly inaccurate. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Electrochemically enhanced antibody immobilization on polydopamine thin film for sensitive surface plasmon resonance immunoassay.

PubMed

Chen, Daqun; Mei, Yihong; Hu, Weihua; Li, Chang Ming

2018-05-15

For sensitive immunoassay, it is essentially important to immobilize antibody on a surface with high density and full retention of their recognition activity. Bio-inspired polydopamine (PDA) thin film has been widely utilized as a reactive coating to immobilize antibody on various surfaces. We herein report that the antibody immobilization capacity of PDA thin film is electrochemically enhanced by applying an oxidative potential to convert the surface catechol group to reactive quinone group. Quantitative surface plasmon resonance (SPR) investigation unveils that upon proper electrochemical oxidization, the antibody loading capacity of PDA film is significantly improved (up to 27%) and is very close to the theoretically maximal capacity of a planar surface if concentrated antibody solution is used. Using prostate-specific antigen (PSA) as a model target, it is further demonstrated that the SPR immunoassay sensitivity is greatly enhanced due to the improved antibody immobilization. This work offers an efficient strategy to enhance the reactivity of PDA film towards nucleophiles, and may also facilitate its immunoassay application among others. Copyright © 2018 Elsevier B.V. All rights reserved.

Nanodiamonds as pH-switchable oxidation and reduction catalysts with enzyme-like activities for immunoassay and antioxidant applications.

PubMed

Chen, T M; Tian, X M; Huang, L; Xiao, J; Yang, G W

2017-10-19

Nanodiamonds (NDs) have recently become a focus of interest from the viewpoints of both science and technology. Their intriguing properties make them suitable as biologically active substrates, in biosensor applications as well as diagnostic and therapeutic biomedical imaging probes. Here, we demonstrate that NDs, as oxidation and reduction catalysts, possess intrinsic enzyme mimetic properties of oxidase, peroxidase and catalase, and these behaviors can be switched by modulating the pH value. NDs not only catalyze the reduction of oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) at acidic pH, but also catalyze the dismutation decomposition of H 2 O 2 to produce O 2 at alkaline pH. It was proposed that the molecular mechanism of their peroxidase-like activity is electron-transfer acceleration, the source of which is likely derived from oxygen containing functional groups on their surface. Based on the color reaction, a nanodiamond-based enzyme linked immunosorbent assay (ELISA) was established for the detection of immunoglobulin G (IgG). Surprisingly, NDs display an excellent antioxidant activity due to the protective effect against H 2 O 2 -induced cellular oxidative damage. These findings make NDs a promising enzyme mimetic candidate and expand their applications in biocatalysis, bioassays and nano-biomedicine.

Sensitive electrochemical immunoassay for 2,4,6-trinitrotoluene based on functionalized silica nanoparticle labels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jun; Liu, Guodong; Wu, Hong

2008-03-03

We present a poly(guanine)-functionalized silica nanoparticle (NP) label-based electrochemical immunoassay for sensitively detecting 2,4,6-trinitrotoluene (TNT). This immunoassay takes advantage of magnetic bead–based platform for competitive displacement immunoreactions and separation, and use electroactive nanoparticles as labels for signal amplification. For this assay, anti-TNT-coated magnetic beads interacted with TNT analog-conjugated poly(guanine)-silica NPs and formed analog-anti-TNT immunocomplexes on magnetic beads. The immunocomplexes coated magnetic beads were exposed to TNT samples, which resulted in displacing the analog conjugated poly(guanine) silica NPs into solution by TNT. In contrast, there are no guanine residues releasing into the solution in the absence of TNT. The reaction solutionmore » was then separated from the magnetic beads and transferred to the electrode surface for electrochemical measurements of guanine oxidation with Ru(bpy)32+ as mediator. The sensitivity of this TNT assay was greatly enhanced through dual signal amplifications: 1) a large amount of guanine residues on silica nanoparticles is introduced into the test solution by displacement immunoreactions and 2) a Ru(bpy)32+-induced guanine catalytic oxidation further enhances the electrochemical signal. Some experimental parameters for the nanoparticle label-based electrochemical immunoassay were studied and the performance of this assay was evaluated. The method is found to be very sensitive and the detection limit of this assay is ~ 0.1 ng mL-1 TNT. The electrochemical immunoassay based on the poly[guanine]-functionalized silica NP label offers a new approach for sensitive detection of explosives.« less

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

PubMed Central

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

2012-01-01

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

Detection of cyclopiazonic acid (CPA) in maize by immunoassay.

PubMed

Maragos, C M; Sieve, K K; Bobell, J

2017-05-01

Cyclopiazonic acid (α-CPA) is a tremorgenic mycotoxin that is commonly produced by certain species of the aspergilli, in particular Aspergillus flavus, which is more widely known for production of the aflatoxins. Despite the fact that α-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for α-CPA. Two mAbs in particular were very sensitive, with IC 50 s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen-immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC 20 to IC 80 ) was from 5 to 28 μg/kg. Recoveries from maize spiked over the range from 2 to 50 μg/kg averaged 88.6 ± 12.6%. Twenty-eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the five samples above the limits of quantitation of both methods, CI-ELISA tended to overestimate CPA content, a difference that we speculate may be due to related metabolites or perhaps "masked" derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for further evaluation of the prevalence of this mycotoxin in maize.

Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

PubMed

Berger, Peter; Lapthorn, Adrian J

2016-01-01

hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

A Retrospective Analysis of Urine Drugs of Abuse Immunoassay True Positive Rates at a National Reference Laboratory.

PubMed

Johnson-Davis, Kamisha L; Sadler, Aaron J; Genzen, Jonathan R

2016-03-01

Urine drug screens are commonly performed to identify drug use or monitor adherence to drug therapy. The purpose of this retrospective study was to evaluate the true positive and false positive rates of one of our in-house urine drug screen panels. The urine drugs of abuse panel studied consists of screening by immunoassay then positive immunoassay results were confirmed by mass spectrometry. Reagents from Syva and Microgenics were used for the immunoassay screen. The screen was performed on a Beckman AU5810 random access automated clinical analyzer. The percent of true positives for each immunoassay was determined. Agreement with previously validated GC-MS or LC-MS-MS confirmatory methods was also evaluated. There were 8,825 de-identified screening results for each of the drugs in the panel, except for alcohol (N = 2,296). The percent of samples that screened positive were: 10.0% for amphetamine/methamphetamine/3,4-methylenedioxy-methamphetamine (MDMA), 12.8% for benzodiazepines, 43.7% for opiates (including oxycodone) and 20.3% for tetrahydrocannabinol (THC). The false positive rate for amphetamine/methamphetamine was ∼14%, ∼34% for opiates (excluding oxycodone), 25% for propoxyphene and 100% for phencyclidine and MDMA immunoassays. Based on the results from this retrospective study, the true positive rate for THC drug use among adults were similar to the rate of illicit drug use in young adults from the 2013 National Survey; however, our positivity rate for cocaine was higher than the National Survey. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MAO enzymes inhibitory activity of new benzimidazole derivatives including hydrazone and propargyl side chains.

PubMed

Can, Özgür Devrim; Osmaniye, Derya; Demir Özkay, Ümide; Sağlık, Begüm Nurpelin; Levent, Serkan; Ilgın, Sinem; Baysal, Merve; Özkay, Yusuf; Kaplancıklı, Zafer Asım

2017-05-05

In the present work, 15 new N'-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzohydrazide (4a-4o) were designed and synthesized. The structures of the synthesized compounds were elucidated using FT-IR, 1 H-NMR, 13 C-NMR, and HRMS spectral data. The inhibitory activity of the compounds 4a-4o against hMAO-A and hMAO-B enzymes was evaluated by using in vitro Amlex Red ® reagent based fluorometric method. Due to lots of high-cost kits including this assay, we determined the ingredients of the kits from the data sheets of several suppliers, and adjusted a protocol by working with various concentrations and volumes of these ingredients. As a result, a fast and sensitive assay was applied as in the commercially available MAO kits with lower costs and clearer ingredients than those of the kits. The enzyme inhibition assay revealed that synthesized compounds have selective inhibition potency against hMAO-B. The compound 4e and 4f displayed IC 50 values of 0.075 μM and 0.136 μM against hMAO-B, respectively. The reference drugs selegiline (IC 50  = 0.040 μM) and rasagiline (IC 50  = 0.066 μM) also displayed a significant inhibition against hMAO-B. The enzyme kinetic study was performed in order to observe the effect of the most active compound 4e on substrate-enzyme relationship and non-competitive inhibition of hMAO-B was determined. Cytotoxicity and genotoxicity studies were carried out and the compound 4e was found as non-cytotoxic and non-genotixic. Theoretical calculation of ADME properties suggested that compound 4e may have a good pharmacokinetic profile. The docking study of compound 4e revealed that there is a strong interaction between the active sites of hMAO-B and analyzed compound. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Surface-Enhanced Raman Scattering-Based Immunoassay Technologies for Detection of Disease Biomarkers

PubMed Central

Smolsky, Joseph; Kaur, Sukhwinder; Hayashi, Chihiro; Batra, Surinder K.; Krasnoslobodtsev, Alexey V.

2017-01-01

Detection of biomarkers is of vital importance in disease detection, management, and monitoring of therapeutic efficacy. Extensive efforts have been devoted to the development of novel diagnostic methods that detect and quantify biomarkers with higher sensitivity and reliability, contributing to better disease diagnosis and prognosis. When it comes to such devastating diseases as cancer, these novel powerful methods allow for disease staging as well as detection of cancer at very early stages. Over the past decade, there have been some advances in the development of platforms for biomarker detection of diseases. The main focus has recently shifted to the development of simple and reliable diagnostic tests that are inexpensive, accurate, and can follow a patient’s disease progression and therapy response. The individualized approach in biomarker detection has been also emphasized with detection of multiple biomarkers in body fluids such as blood and urine. This review article covers the developments in Surface-Enhanced Raman Scattering (SERS) and related technologies with the primary focus on immunoassays. Limitations and advantages of the SERS-based immunoassay platform are discussed. The article thoroughly describes all components of the SERS immunoassay and highlights the superior capabilities of SERS readout strategy such as high sensitivity and simultaneous detection of a multitude of biomarkers. Finally, it introduces recently developed strategies for in vivo biomarker detection using SERS. PMID:28085088

Electrophoretic build-up of multi nanoparticle array for a highly sensitive immunoassay

PubMed Central

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

2012-01-01

One of the challenges in shrinking immunoassays to smaller sizes is to immobilize the biological molecules to nanometer-scaled spots. To overcome this complication, we have employed a particle-based immunoassay to create a nanostructured platform with a regular array of sensing elements. The technique makes use of an electrophoretic particle entrapment system (EPES) to immobilize nanoparticles that are coated with biological reagents into wells using a very small trapping potential. To provide useful information for controlling the trapping force and optimal design of the nanoarray, electrophoretic trapping of a nanoparticle was modeled numerically. The trapping efficiency, defined as the fraction of wells occupied by a single particle, was 91%. The performance of the array was demonstrated with a competitive immunoassay for a small molecule analyte, 3-phenoxybenzoic acid (214.2 g mole−1). The limit of detection determined with a basic fluorescence microscope was 0.006 μg l−1 (30 pM); this represented a sixteen-fold improvement in sensitivity compared to a standard 96-well plate-based ELISA; the improvement was attributed to the small size of the sample volume and the presence of light diffraction among factors unique to this structure. The EPES/nanoarray system promises to offer a new standard in applications that require portable, point-of-care and real-time monitoring with high sensitivity. PMID:23021853

Pooled nucleic acid testing increases the diagnostic yield of acute HIV infections in a high-risk population compared to 3rd and 4th generation HIV enzyme immunoassays.

PubMed

Krajden, Mel; Cook, Darrel; Mak, Annie; Chu, Ken; Chahil, Navdeep; Steinberg, Malcolm; Rekart, Michael; Gilbert, Mark

2014-09-01

We compared a 3rd generation (gen) and two 4th gen HIV enzyme immunoassays (EIA) to pooled nucleic acid testing (PNAT) for the identification of pre- and early seroconversion acute HIV infection (AHI). 9550 specimens from males >18 year from clinics attended by men who have sex with men were tested by Siemens ADVIA Centaur(®) HIV 1/O/2 (3rd gen) and HIV Combo (4th gen), as well as by Abbott ARCHITECT(®) HIV Ag/Ab Combo (4th gen). Third gen non-reactive specimens were also tested by Roche COBAS(®) Ampliprep/COBAS® TaqMan HIV-1 Test v.2 in pools of 24 samples. Sensitivity and specificity of the three EIAs for AHI detection were compared. 7348 persons contributed 9435 specimens and had no evidence of HIV infection, 79 (94 specimens) had established HIV infection, 6 (9 specimens) had pre-seroconversion AHI and 9 (12 specimens) had early seroconversion AHI. Pre-seroconversion AHI cases were not detected by 3rd gen EIA, whereas 2/6 (33.3%) were detected by Siemens 4th gen, 4/6 (66.7%) by Abbott 4th gen and 6/6 (100%) by PNAT. All three EIAs and PNAT detected all individuals with early seroconversion AHI. Overall sensitivity/specificity for the EIAs relative to WB or NAT resolved infection status was 93.6%/99.9% for Siemens 3rd gen, 95.7%/99.7% for Siemens 4th gen and 97.9%/99.2% for Abbott 4th gen. While both 4th gen EIAs demonstrated improved sensitivity for AHI compared to 3rd gen EIA, PNAT identified more AHI cases than either 4th gen assay. PNAT is likely to remain a useful strategy to identify AHI in high-risk populations. Copyright © 2014 Elsevier B.V. All rights reserved.

Manual-slide-engaged paper chip for parallel SERS-immunoassay measurement of clenbuterol from swine hair.

PubMed

Zheng, Tingting; Gao, Zhigang; Luo, Yong; Liu, Xianming; Zhao, Weijie; Lin, Bingcheng

2016-02-01

Clenbuterol (CL), as a feed additive, has been banned in many countries due to its potential threat to human health. In detection of CL, a fast, low-cost technique with high accuracy and specificity would be ideal for its administrative on-field inspections. Among the attempts to pursue a reliable detection tool of CL, a technique that combines surface enhanced Raman spectroscopy (SERS) and immunoassay, is close to meet the requirements as above. However, multiple steps of interactions between CL analyte, antibody, and antigen are involved in this method, and under conventional setup, the operation of SERS/immunoassay were unwieldy. In this paper, to facilitate a more manageable sample manipulation for SERS-immunoassay measurement, a 3D paper chip was suggested. A switch-on-chip multilayered (abbreviated as SoCM-) microfluidic paper-based analysis device (μPad) was fabricated to provide operators with manual switches on the interactions between different microfluids. Besides, on a detection slip we made on the main body of our SoCM-μPad, antigen was anchored in pattern. With this architecture, multistep interactions between the CL analyte in swine hair extract and the SERS probe-modified antibody and antigen, were managed for on-chip SERS-immunoassay detection. This would be very attractive for fast, cheap, accurate, and on-site specific detection of CL from real samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

PubMed

Koller, E; Wolfbeis, O S

1984-11-15

A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

[Immunoassay for matrix metalloproteinase-9 in the tear film of patients with pseudoexfoliation syndrome - a pilot study].

PubMed

Zimmermann, N; Erb, C

2013-08-01

Matrix-metalloproteinases (MMPs) are proteolytic enzymes released by irritated epithelial cells of the ocular surface. It has been established that the subtype MMP-9 can serve as an inflammatory marker within the tear film. MMP-9 is also attributed to have an effect on the PEX-glaucoma development. Recently, a rapid immunoassay for detection of MMP-9 in the tear film was developed to estimate inflammatory extent during dry eye disease. The aim of this study was to analyse the MMP-9 concentration in tear film in PEX-syndrome. In addition, an assessment of the feasibility, reliability and readability of the test was done. We randomly selected 10 patients with PEX-syndrome and 10 healthy control patients and measured tear film MMP-9 of one eye with the RPS InflammaDry Detector™ (Rapid Pathogen Screening Inc., USA). We detected increased levels of MMP-9 in tear film in PEX-syndrome. 80 % of the PEX-patients and 20 % of the controls showed a positive test result (>or= 40 ng/mL MMP-9) indicating a test specificity and sensitivity of 80 %. This corresponds approximately to the published values for the dry eye (sensitivity 87 %, specificity: 92 %). The performance of the test is simple. The patients tolerated the inclusion of the test strips well. However, it is difficult to estimate whether enough tear film was used and in many cases, the intensity of the "indicator line" was weak. The rapid MMP-9-immunoassay is a novel, meaningful approach for the detection of inflammatory activity of the ocular surface. We have shown an up-regulation of the non-specific inflammatory marker MMP-9 in tear film in PEX-syndrome and suggest an association with a tear film disorder. However, an improvement in the estimation of the amount of collected tears and readability is desirable. Georg Thieme Verlag KG Stuttgart · New York.

Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

NASA Astrophysics Data System (ADS)

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

2004-06-01

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

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

PubMed

Bose, Debosreeta; Sarkar, Deboleena; Chattopadhyay, Nitin

2010-01-01

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

Multi-commutated fluorometric optosensor for the determination of citrinin in rice and red yeast rice supplements.

PubMed

Jiménez-López, J; Llorent-Martínez, E J; Ortega-Barrales, P; Ruiz-Medina, A

2014-01-01

Citrinin is a toxic secondary metabolite first isolated from Penicillium citrinum, although is also produced by other species of Penicillium and Aspergillus. It has highly toxic, mutagenic, teratogenic and carcinogenic properties and is often found in crops, vegetables and fruit. To our knowledge there is no specific legislation on maximum levels permitted for citrinin, so no official analytical method is currently available for its determination. Our laboratory developed a fluorometric flow-through optosensor using Sephadex SPC-25 as solid support. Multi-commutated flow injection analysis was used for the construction of the manifold and for handling solutions. In this way, we minimised waste generation and human intervention, which are critical aspects when dealing with highly toxic compounds such as citrinin. The optimum excitation/emission wavelengths were set at 330/494 nm; the calibration curve was linear in the concentration range 35-900 ng ml⁻¹. A detection limit of 10.5 ng ml⁻¹ and relative standard deviations (RSDs) lower than 3% were obtained. The developed optosensor was applied to the determination of citrinin in rice and dietary supplements containing red yeast rice.

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review.

PubMed

Xu, Zhen-Lin; Shen, Yu-Dong; Beier, Ross C; Yang, Jin-Yi; Lei, Hong-Tao; Wang, Hong; Sun, Yuan-Ming

2009-08-11

Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional 'trial and error' method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody-antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

PubMed

Szkola, A; Linares, E M; Worbs, S; Dorner, B G; Dietrich, R; Märtlbauer, E; Niessner, R; Seidel, M

2014-11-21

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

Cross-Reactivity of Pantoprazole with Three Commercial Cannabinoids Immunoassays in Urine.

PubMed

Gomila, Isabel; Barceló, Bernardino; Rosell, Antonio; Avella, Sonia; Sahuquillo, Laura; Dastis, Macarena

2017-11-01

Pantoprazole is a frequently prescribed proton pump inhibitor (PPI) commonly utilized in the management of gastrointestinal symptoms. Few substances have proved to cause a false-positive cannabinoid urine screen. However, a case of false-positive urine cannabinoid screen in a patient who received a pantoprazole dose has been recently published. The purpose of this study was to determine the potential cross-reactivity of pantoprazole in the cannabinoid immunoassays: Alere Triage® TOX Drug Screen, KIMS® Cannabinoids II and DRI® Cannabinoids Assay. Drug-free urine to which pantoprazole was added up to 12,000 μg/mL produced negative results in the DRI® Cannabinoids and KIMS® Cannabinoids II. Alere Triage® TOX Drug Screen assay gave positive results at pantoprazole concentrations higher than 1,000 μg/mL. Urine samples from 8 pediatric patients were collected at the beginning of their pantoprazole treatment. Alere Triage® TOX Drug Screen assay produced positive test results in all patient samples and KIMS® Cannabinoids II immunoassay produced positive test results in one patient sample. None patient sample gave a false-positive result when analyzed by the DRI® Cannabinoids Assay. Our findings demonstrate that some cannabinoids immunoassays are susceptible to cross-reaction errors resulting from the presence in urine of pantoprazole and the resulting metabolism of the parent drug. Clinicians should be aware of the possibility of false-positive results for cannabinoids after a pantoprazole treatment. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fluorescence polarization immunoassays for rapid, accurate and sensitive determination of mycotoxins

USDA-ARS?s Scientific Manuscript database

Fluorescence polarization immunoassay (FPIA) is a type of homogeneous assay. For low molecular weight antigens, such as mycotoxins, it is based on the competition between an unlabeled antigen and its fluorescent-labeled derivative (tracer) for an antigen-specific antibody. The antigen content is det...

IMMUNOASSAY METHOD FOR THE DETERMINATION OF PENTACHLOROPHENOL IN SOIL AND SEDIMENT

EPA Science Inventory

The journal article describes the use of a prototype immunoassay method for the determination of pentacholorphenol (PCP) in soil and sediment. PCP was used as a pesticide and wood preservative and is not currently available to the general public. The paper stresses the importan...

Target-induced displacement reaction accompanying cargo release from magnetic mesoporous silica nanocontainers for fluorescence immunoassay.

PubMed

Tang, Dianping; Liu, Bingqian; Niessner, Reinhard; Li, Peiwu; Knopp, Dietmar

2013-11-05

A new fluorescence immunoassay strategy based on a target-induced displacement reaction with cargo release from protein-gated carbohydrate-functionalized magnetic mesoporous silica nanoparticles (MMSN) was developed for sensitive detection of small molecular mycotoxins (aflatoxin B1, AFB1 used in this case). To construct such an assay system, MMSN was initially functionalized with mannose-terminated silanes, then capped with biotinylated concanavalin A (Con A) entrapped rhodamine B (RB) within the pores through the carbohydrate-protein interaction, and then biotinylated monoclonal anti-AFB1 capture antibody was conjugated to Con A-functionalized MMSN by the streptavidin-biotin chemistry. Gold nanoparticles (AuNP) heavily functionalized with invertase and bovine serum albumin-AFB1 conjugate were utilized as the trace tag. With AFB1 introduction, a competitive immunoreaction for the immobilized anti-AFB1 antibody on the MMSN was started between target analyte and the labeled AFB1 on the AuNP. Accompanied by AuNP, the carried invertase hydrolyzed sucrose in glucose and fructose. The generated glucose competed with the mannose for Con A and displaced the Con A-antibody complex from the MMSN, resulting in the opening of molecular gates owing to the uncapping of MMSN, thereby the entrapped RB could release from the pores. The released RB could be quantitatively determined by a fluorometer. Under optimal conditions, the fluorescence intensity decreased with the increasing AFB1 concentration in the range from 0.01 to 5 ng mL(-1) with a detection limit (LOD) of 8 pg mL(-1) at the 3sblank criterion. Intra- and interbatch assay precisions were lower than 9 and 9.5% (CV), respectively. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of naturally contaminated peanut samples between the fluorescence immunoassay and a commercialized enzyme

Fluorometric Measurement of Pyridine Nucleotide Reduction in the Giant Axon of the Squid

PubMed Central

Doane, Marshall G.

1967-01-01

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1–2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25–50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10–20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon. PMID:4384698

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

PubMed

Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

2016-06-21

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

PubMed

Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Liu, J Q; Zhang, Y; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S

2014-11-15

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample. Copyright © 2014 Elsevier B.V. All rights reserved.

Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests

PubMed Central

Santos, Fred Luciano Neves; de Souza, Wayner Vieira; da Silva Barros, Michelle; Nakazawa, Mineo; Krieger, Marco Aurélio; de Miranda Gomes, Yara

2016-01-01

There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic. PMID:26976886

Lateral flow immunoassay for the rapid detection of citrus tristeza virus

USDA-ARS?s Scientific Manuscript database

A lateral flow methodology was developed using gold nanoparticles for rapid detection of Citrus tristeza virus (CTV). The test strip was based on a sandwich immunoassay and could be accomplished within 10 minutes. A sample was considered negative for CTV when only the control line appeared; whereas,...

Integration of minisolenoids in microfluidic device for magnetic bead-based immunoassays

NASA Astrophysics Data System (ADS)

Liu, Yan-Jun; Guo, Shi-Shang; Zhang, Zhi-Ling; Huang, Wei-Hua; Baigl, Damien; Chen, Yong; Pang, Dai-Wen

2007-10-01

Microfluidic devices with integrated minisolenoids, microvalves, and channels have been fabricated for fast and low-volume immunoassay using superparamagnetic beads and well-known surface bioengineering protocols. A magnetic reaction area can be formed in the microchannel, featuring a high surface-to-volume ratio and low diffusion distances for the reagents to the bead surface. Such a method has the obvious advantage of easy implementation at low cost. Moreover, the minisolenoids can be switched on or off and the magnetic field intensity can be tuned on demand. Fluids can be manipulated by controlling the integrated air-pressure-actuated microvalves. Accordingly, magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic device automatically in longitudinal mode. With a sample consumption of 0.5μl and a total assay time of less than 15min, goat immunoglobulin G was detected and the method exhibited a detection limit of 4.7ng/ml.

Immunoassay and Nb2 lymphoma bioassay prolactin levels and mammographic density in premenopausal and postmenopausal women the Nurses' Health Studies.

PubMed

Rice, Megan S; Tworoger, Shelley S; Bertrand, Kimberly A; Hankinson, Susan E; Rosner, Bernard A; Feeney, Yvonne B; Clevenger, Charles V; Tamimi, Rulla M

2015-01-01

Higher circulating prolactin levels have been associated with higher percent mammographic density among postmenopausal women in some, but not all studies. However, few studies have examined associations with dense area and non-dense breast area breast or considered associations with prolactin Nb2 lymphoma cell bioassay levels. We conducted a cross-sectional study among 1,124 premenopausal and 890 postmenopausal women who were controls in breast cancer case-control studies nested in the Nurses' Health Study (NHS) and NHSII. Participants provided blood samples in 1989-1990 (NHS) or 1996-1999 (NHSII) and mammograms were obtained from around the time of blood draw. Multivariable linear models were used to assess the associations between prolactin levels (measured by immunoassay or bioassay) with percent density, dense area, and non-dense area. Among 1,124 premenopausal women, percent density, dense area, and non-dense area were not associated with prolactin immunoassay levels in multivariable models (p trends = 0.10, 0.18, and 0.69, respectively). Among 890 postmenopausal women, those with prolactin immunoassay levels in the highest versus lowest quartile had modestly, though significantly, higher percent density (difference = 3.01 percentage points, 95 % CI 0.22, 5.80) as well as lower non-dense area (p trend = 0.02). Among women with both immunoassay and bioassay levels, there were no consistent differences in the associations with percent density between bioassay and immunoassay levels. Postmenopausal women with prolactin immunoassay levels in the highest quartile had significantly higher percent density as well as lower non-dense area compared to those in the lowest quartile. Future studies should examine the underlying biologic mechanisms, particularly for non-dense area.

Fabrication and Evaluation of Microfluidic Immunoassay Devices with Antibody-Immobilized Microbeads Retained in Porous Hydrogel Micropillars.

PubMed

Kasama, Toshihiro; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

2017-01-01

Due to the inherent characteristics including confinement of molecular diffusion and high surface-to-volume ratio, microfluidic device-based immunoassay has great advantages in cost, speed, sensitivity, and so on, compared with conventional techniques such as microtiter plate-based ELISA, latex agglutination method, and lateral flow immunochromatography. In this paper, we explain the detection of C-reactive protein as a model antigen by using our microfluidic immunoassay device, so-called immuno-pillar device. We describe in detail how we fabricated and used the immuno-pillar devices.