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.

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.

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

ATimer-Actuated, Immunoassay Cassette for Detecting Molecular Markers in Oral Fluids

PubMed Central

Liu, Changchun; Qiu, Xianbo; Ongagna, Serge; Chen, Dafeng; Chen, Zongyuan; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L.A.M.; Bau, Haim H.

2009-01-01

An inexpensive, hand-held, point-of-care, disposable, self-contained, immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting, phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource poor countries, where funds and trained personnel are in short supply. PMID:19255658

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

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.

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...

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.

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.

Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.

PubMed

Pape, B E; Cary, P L; Clay, L C; Godolphin, W

1983-01-01

Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles.

PubMed

Wacker, Ron; Ceyhan, Buelent; Alhorn, Petra; Schueler, Dirk; Lang, Claus; Niemeyer, Christof M

2007-06-01

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.

[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.

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

Stimuli-Responsive Reagent System for Enabling Microfluidic Immunoassays with Biomarker Purification and Enrichment

PubMed Central

2015-01-01

Immunoassays have been translated into microfluidic device formats, but significant challenges relating to upstream sample processing still limit their applications. Here, stimuli-responsive polymer–antibody conjugates are utilized in a microfluidic immunoassay to enable rapid biomarker purification and enrichment as well as sensitive detection. The conjugates were constructed by covalently grafting poly(N-isopropylacrylamide) (PNIPAAm), a thermally responsive polymer, to the lysine residues of anti-prostate specific antigen (PSA) Immunoglobulin G (IgG) using carbodiimide chemistry via the polymer end-carboxylate. The antibody-PNIPAAm (capture) conjugates and antibody-alkaline phosphatase (detection) conjugates formed sandwich immunocomplexes via PSA binding in 50% human plasma. The complexes were loaded into a recirculating poly(dimethylsiloxane) microreactor, equipped with micropumps and transverse flow features, for subsequent separation, enrichment, and quantification. The immunocomplexes were captured by heating the solution to 39 °C, mixed over the transverse features for 2 min, and washed with warm buffer. In one approach, the assay utilized immunocomplex solution that was contained in an 80 nL microreactor, which was loaded with solution at room temperature and subsequently heated to 39 °C. The assay took 25 min and resulted in 37 pM PSA limit of detection (LOD), which is comparable to a plate ELISA employing the same antibody pair. In another approach, the microreactor was preheated to 39 °C, and immunocomplex solution was flowed through the reactor, mixed, and washed. When the specimen volume was increased to 7.5 μL by repeating the capture process three times, the higher specimen volume led to immunocomplex enrichment within the microreactor. The resulting assay LOD was 0.5 pM, which is 2 orders of magnitude lower than the plate ELISA. Both approaches generate antigen specific signal over a clinically significant range. The sample processing

Resolution of an apparent hook effect in Roche partner DRI oxycodone immunoassay.

PubMed

Colón-Franco, Jessica M; Cox, Elbert T; Crosby, David B; Dawling, Sheila

2013-01-01

A presumed hook effect in the semiquantitative DRI Oxycodone immunoassay, OXY3S (Cobas Integra, Roche Diagnostics), was investigated in 14 urine samples with gas chromatography/mass spectrometry (GC-MS) >10,000 ng/mL but OXY3S <1,000 ng/mL. These samples included the index case, a false-negative OXY3S result with >75,000 ng/mL oxycodone + oxymorphone by GC-MS confirmation. Patient samples needed 2- to 16-fold dilution to obtain the correct OXY3S response. The OXY3S test did not hook at high-spiked concentrations of oxycodone, oxymorphone or oxymorphone-3β-d-glucuronide in drug-free urine. The OXY3S test parameters were replicated in a development channel on the Cobas using DRI Reagents (Microgenics, CA, USA) and were subsequently modified. Delayed sample addition or doubling of Reagent 1 (R1: antibody/substrate/co-factor) yielded maximal immunoassay response (>10,000 ng/mL) in 12 of 14 and 14 of 14 undiluted patient samples, respectively. Supplementation of R1 with substrate alone did not correctly recover oxycodone from any of the samples, while co-factor supplementation resulted a maximal OXY3S response in 13 of 14 samples. The remaining (index) sample could only be corrected by supplemental R1. The semiquantitative utility of the DRI Oxycodone assay is questionable. Although the precise cause of the under-recovery could not be determined, the modification presented permits reliable oxycodone determination at the high concentrations frequently seen in clinical urine samples.

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

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

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

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.

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.

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.

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 Portable Analyzer for Pouch-Actuated, Immunoassay Cassettes

PubMed Central

Qiu, Xianbo; Liu, Changchun; Mauk, Michael G.; Hart, Robert W.; Chen, Dafeng; Qiu, Jing; Kientz, Terry; Fiene, Jonathan; Bau, Haim H.

2011-01-01

A portable, small footprint, light, general purpose analyzer (processor) to control the flow in immunoassay cassettes and to facilitate the detection of test results is described. The durable analyzer accepts disposable cassettes that contain pouches and reaction chambers for various unit operations such as hydration of dry reagents, stirring, and incubation. The analyzer includes individually controlled, linear actuators to compress the pouches in the cassette, which facilitates the pumping and mixing of sample and reagents, and to close diaphragm-based valves for flow control. The same types of actuators are used to compress pouches and actuate valves. The analyzer also houses a compact OEM scanner/reader to excite fluorescence and detect emission from labels. The analyzer is hydraulically isolated from the cassette, reducing the possibility of cross-contamination. The analyzer facilitates programmable, automated execution of a sequence of operations such as pumping and valving in a timely fashion, reducing the level of expertise required from the operator and the possibility for errors. The analyzer’s design is modular and expandable to accommodate cassettes of various complexities and additional functionalities. In this paper, the utility of the analyzer has been demonstrated with the execution of a simple, consecutive, lateral flow assay of a model biological system and the test results were detected with up converting phosphor labels that are excited at infrared frequencies and emit in the visible spectrum. PMID:22125359

Rapid extraction, identification and quantification of drugs of abuse in hair by immunoassay and ultra-performance liquid chromatography tandem mass spectrometry.

PubMed

Pichini, Simona; Gottardi, Massimo; Marchei, Emilia; Svaizer, Fiorenza; Pellegrini, Manuela; Rotolo, Maria Concetta; Algar, Oscar García; Pacifici, Roberta

2014-05-01

Drug testing in hair is a unique analysis in pharmacotoxicology for establishing a past repeated history of consumption or passive exposure to psychotropic substances. A rather lengthy sample treatment is usually required before parent drugs and eventual metabolites are amenable to quali-quantitative analysis. We evaluated a high throughput screening and confirmation analysis of drugs of abuse in hair by immunoassay and a validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) after applying a rapid digestion of the keratin matrix with VMA-T reagent before screening assay and M3 reagent before confirmatory analysis. Samples digestion with VMA-T reagent and immunometric screening analysis of hair calibrators, controls and clinical samples for a total of 150 samples was completed in 4 h. No false-positive and -negative results were found for the control material. UPLC-MS/MS analysis confirmed all of the 31 adult hair samples positive to the screening test using internationally established cut-offs, and identified and quantified drugs of abuse in 32 pediatric hair samples, applying lower limits of quantification from 0.01 to 0.1 ng analyte per mg hair. Analytical recovery was between 70.9% and 100.7%. Intra- and inter-assay imprecision and inaccuracy were always lower than 10%. Rapid extraction, identification and quantification of drugs of abuse in hair by immunoassay and UPLC-MS/MS was tested for its feasibility in clinical samples and provided excellent results for rapid and effective drug testing in hair in epidemiological studies.

Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

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

Shi, Tujin; Qian, Weijun

2013-02-01

Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

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.

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.

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2013-01-01

Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent

Primary care requests for anaemia chemistry tests in Spain: potential iron, transferrin and folate over-requesting.

PubMed

Salinas, Maria; López-Garrigós, Maite; Flores, Emilio; Leiva-Salinas, Carlos

2017-09-01

To study the regional variability of requests for anaemia chemistry tests in primary care in Spain and the associated economic costs of potential over-requesting. Requests for anaemia tests were examined in a cross-sectional study. Clinical laboratories from different autonomous communities (AACCs) were invited to report on primary care anaemia chemistry tests requested during 2014. Demand for iron, ferritin, vitamin B12 and folate tests per 1000 inhabitants and the ratios of the folate/vitamin B12 and transferrin/ferritin requests were compared between AACCs. We also calculated reagent costs and the number of iron, transferrin and folate tests and the economic saving if every AACC had obtained the results achieved by the AACC with best practice. 110 laboratories participated (59.8% of the Spanish population). More than 12 million tests were requested, resulting in reagent costs exceeding €16.5 million. The serum iron test was the most often requested, and the ferritin test was the most costly (over €7 million). Close to €4.5 million could potentially have been saved if iron, transferrin and folate had been appropriately requested (€6 million when extrapolated to the whole Spanish population). The demand for and expenditure on anaemia chemistry tests in primary care in Spain is high, with significant regional differences between different AACCs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

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.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

PubMed

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Multi-center Performance Evaluations of Tacrolimus and Cyclosporine Electrochemiluminescence Immunoassays in the Asia-Pacific Region

PubMed Central

Qin, Xuzhen; Rui, Jianzhong; Xia, Yong; Mu, Hong; Song, Sang Hoon; Raja Aziddin, Raja Elina; Miles, Gabrielle; Sun, Yuli

2018-01-01

Background The immunosuppressant drugs (ISDs), tacrolimus and cyclosporine, are vital for solid organ transplant patients to prevent rejection. However, toxicity is a concern, and absorption is highly variable across patients; therefore, ISD levels need to be precisely monitored. In the Asia-Pacific (APAC) region, tacrolimus and cyclosporine concentrations are typically measured using immunoassays. The objective of this study was to assess the analytical performance of Roche Elecsystacrolimus and cyclosporinee electrochemiluminescence immunoassays (ECLIAs). Methods This evaluation was performed in seven centers across China, South Korea, and Malaysia. Imprecision (repeatability and reproducibility), assay accuracy, and lot-to-lot reagent variability were tested. The Elecsys ECLIAs were compared with commercially available immunoassays (Architect, Dimension, and Viva-E systems) using whole blood samples from patients with various transplant types (kidney, liver, heart, and bone marrow). Results Coefficients of variation for repeatability and reproducibility were ≤5.4% and ≤12.4%, respectively, for the tacrolimus ECLIA, and ≤5.1% and ≤7.3%, respectively, for the cyclosporine ECLIA. Method comparisons of the tacrolimus ECLIA with Architect, Dimension, and Viva-E systems yielded slope values of 1.01, 1.14, and 0.897, respectively. The cyclosporine ECLIA showed even closer agreements with the Architect, Dimension, and Viva-E systems (slope values of 1.04, 1.04, and 1.09, respectively). No major differences were observed among the different transplant types. Conclusions The tacrolimus and cyclosporine ECLIAs demonstrated excellent precision and close agreement with other immunoassays tested. These results show that both assays are suitable for ISD monitoring in an APAC population across a range of different transplant types. PMID:29214751

Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination.

PubMed

Chiem, N H; Harrison, D J

1998-03-01

A glass microchip is described in which reagents and serum samples for competitive immunoassay of serum theophylline can be mixed, reacted, separated, and analyzed. The device functions as an automated microfluidic immunoassay system, creating a lab-on-a-chip. Electroosmotic pumping was used to control first the mixing of 50-fold-diluted serum sample with labeled theophylline tracer in a 1:1 ratio, followed by 1:1 mixing and reaction with anti-theophylline antibody. The 51-nL on-chip mixer gave the same concentration as dilution performed off-chip, within 3%. A 100-pL plug of the reacted solution was then injected into an electrophoresis separation channel integrated within the same chip. Measurements of free and bound tracer by fluorescence detection gave linear calibration curves of signal vs log[theophylline] between 0 and 40 mg/L, with a slope of 0.52 +/- 0.03 and an intercept of -0.04 +/- 0.04 after a 90-s reaction time. A detection limit of 0.26 mg/L in serum (expressed before the dilution step, actual concentration of 1.3 micrograms/L at the detector) was obtained. Recovery values were 107% +/- 8% for 15 mg/L serum samples.

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

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.

Critical ligand binding reagent preparation/selection: when specificity depends on reagents.

PubMed

Rup, Bonita; O'Hara, Denise

2007-05-11

Throughout the life cycle of biopharmaceutical products, bioanalytical support is provided using ligand binding assays to measure the drug product for pharmacokinetic, pharmacodynamic, and immunogenicity studies. The specificity and selectivity of these ligand binding assays are highly dependent on the ligand binding reagents. Thus the selection, characterization, and management processes for ligand binding reagents are crucial to successful assay development and application. This report describes process considerations for selection and characterization of ligand binding reagents that are integral parts of the different phases of assay development. Changes in expression, purification, modification, and storage of the ligand binding reagents may have a profound effect on the ligand binding assay performance. Thus long-term management of the critical ligand binding assay reagents is addressed including suggested characterization criteria that allow ligand binding reagents to be used in as consistent a manner as possible. Examples of challenges related to the selection, modification, and characterization of ligand binding reagents are included.

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.

Single-bead arrays for fluorescence-based immunoassays on capillary-driven microfluidic chips

NASA Astrophysics Data System (ADS)

Temiz, Yuksel; Lim, Michel; Delamarche, Emmanuel

2016-03-01

We report a concept for the simple fabrication of easy-to-use chips for immunoassays in the context of point-of-care diagnostics. The chip concept comprises mainly three features: (1) the efficient integration of reagents using beads functionalized with receptors, (2) the generation of capillary-driven liquid flows without using external pumps, and (3) a high-sensitivity detection of analytes using fluorescence microscopy. We fabricated prototype chips using dry etching of Si wafers. 4.5-μm-diameter beads were integrated into hexagonal arrays by sedimentation and removing the excess using a stream of water. We studied the effect of different parameters and showed that array occupancies from 30% to 50% can be achieved by pipetting a 250 nL droplet of 1% bead solution and allowing the beads sediment for 3 min. Chips with integrated beads were sealed using a 50-μm-thick dry-film resist laminated at 45 °C. Liquids pipetted to loading pads were autonomously pulled by capillary pumps at a rate of 0.35 nL s-1 for about 30 min. We studied ligand-receptor interactions and binding kinetics using time-lapse fluorescence microscopy and demonstrated a 5 pM limit of detection (LOD) for an anti-biotin immunoassay. As a clinically-relevant example, we implemented an immunoassay to detect prostate specific antigen (PSA) and showed an LOD of 108 fM (i.e. 3.6 pg mL-1). While a specific implementation is provided here for the detection of PSA, we believe that combining capillary-driven microfluidics with arrays of single beads and fluorescence readout to be very flexible and sufficiently sensitive for the detection of other clinically-relevant analytes.

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.

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

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.

Evaluation of Beckman Coulter DxI 800 immunoassay system using clinically oriented performance goals.

PubMed

Akbas, Neval; Schryver, Patricia G; Algeciras-Schimnich, Alicia; Baumann, Nikola A; Block, Darci R; Budd, Jeffrey R; Gaston, S J Stephen; Klee, George G

2014-11-01

We evaluated the analytical performance of 24 immunoassays using the Beckman Coulter DxI 800 immunoassay systems at Mayo Clinic, Rochester, MN for trueness, precision, detection limits, linearity, and consistency (across instruments and reagent lots). Clinically oriented performance goals were defined using the following methods: trueness-published desirable accuracy limits, precision-published desirable biologic variation; detection limits - 0.1 percentile of patient test values, linearity - 50% of total error, and consistency-percentage test values crossing key decision points. Local data were collected for precision, linearity, and consistency. Data were provided by Beckman Coulter, Inc. for trueness and detection limits. All evaluated assays except total thyroxine were within the proposed goals for trueness. Most of the assays met the proposed goals for precision (86% of intra-assay results and 75% of inter-assay results). Five assays had more than 15% of the test results below the minimum detection limits. Carcinoembryonic antigen, total thyroxine and free triiodothyronine exceeded the proposed goals of ±6.3%, ±5% and ±5.7% for dilution linearity. All evaluated assays were within the proposed goals for instrument consistency. Lot-to-lot consistency results for cortisol, ferritin and total thyroxine exceeded the proposed goals of 3.3%, 11.4% and 7% at one medical decision level, while vitamin B12 exceeded the proposed goals of 5.2% and 3.8% at two decision levels. The Beckman Coulter DxI 800 immunoassay system meets most of these proposed goals, even though these clinically focused performance goals represent relatively stringent limits. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Adaptable Detection Strategies in Membrane-Based Immunoassays: Calibration-Free Quantitation with Surface-Enhanced Raman Scattering Readout.

PubMed

Skuratovsky, Aleksander; Soto, Robert J; Porter, Marc D

2018-06-19

This paper presents a method for immunometric biomarker quantitation that uses standard flow-through assay reagents and obviates the need for constructing a calibration curve. The approach relies on a nitrocellulose immunoassay substrate with multiple physical addresses for analyte capture, each modified with different amounts of an analyte-specific capture antibody. As such, each address generates a distinctly different readout signal that is proportional to the analyte concentration in the sample. To establish the feasibility of this concept, equations derived from antibody-antigen binding equilibrium were first applied in modeling experiments. Next, nitrocellulose membranes with multiple capture antibody addresses were fabricated for detection of a model analyte, human Immunoglobulin G (hIgG), by a heterogeneous sandwich immunoassay using antibody-modified gold nanoparticles (AuNPs) as the immunolabel. Counting the number of colored capture addresses visible to the unassisted eye enabled semiquantitative hIgG determination. We then demonstrated that, by leveraging the localized surface plasmon resonance of the AuNPs, surface-enhanced Raman spectroscopy (SERS) can be used for quantitative readout. By comparing the SERS signal intensities from each capture address with values predicted using immunoassay equilibrium theory, the concentration of hIgG can be determined (∼30% average absolute deviation) without reference to a calibration curve. This work also demonstrates the ability to manipulate the dynamic range of the assay over ∼4 orders of magnitude (from 2 ng mL -1 to 10 μg mL -1 ). The potential prospects in applying this concept to point-of-need diagnostics are also discussed.

The microassay on a card: A rugged, portable immunoassay

NASA Technical Reports Server (NTRS)

Kidwell, David

1991-01-01

The Microassay on a Card (MAC) is a portable, hand-held, non-instrumental immunoassay that can test for the presence of a wide variety of substances in the environment. The MAC is a simple device to use. A drop of test solution is placed on one side of the card and within five minutes a color is developed on the other side in proportion to the amount of substance in the test solution, with sensitivity approaching 10 ng/ml. The MAC is self-contained and self-timed; no reagents or timing is necessary. The MAC may be configured with multiple wells to provide simultaneous testing for multiple species. As envisioned, the MAC will be employed first as an on-site screen for drugs of abuse in urine or saliva. If the MAC can be used as a screen of saliva for drugs of abuse, it could be applied to driving while intoxicated, use of drugs on the job, or testing of the identity of seized materials. With appropriate modifications, the MAC also could be used to test for environmental toxins or pollutants.

Development of an ultra-high sensitive immunoassay with plasma biomarker for differentiating Parkinson disease dementia from Parkinson disease using antibody functionalized magnetic nanoparticles.

PubMed

Yang, Shieh-Yueh; Chiu, Ming-Jang; Lin, Chin-Hsien; Horng, Herng-Er; Yang, Che-Chuan; Chieh, Jen-Jie; Chen, Hsin-Hsien; Liu, Bing-Hsien

2016-06-08

It is difficult to discriminate healthy subjects and patients with Parkinson disease (PD) or Parkinson disease dementia (PDD) by assaying plasma α-synuclein because the concentrations of circulating α-synuclein in the blood are almost the same as the low-detection limit using current immunoassays, such as enzyme-linked immunosorbent assay. In this work, an ultra-sensitive immunoassay utilizing immunomagnetic reduction (IMR) is developed. The reagent for IMR consists of magnetic nanoparticles functionalized with antibodies against α-synuclein and dispersed in pH-7.2 phosphate-buffered saline. A high-Tc superconducting-quantum-interference-device (SQUID) alternative-current magnetosusceptometer is used to measure the IMR signal of the reagent due to the association between magnetic nanoparticles and α-synuclein molecules. According to the experimental α-synuclein concentration dependent IMR signal, the low-detection limit is 0.3 fg/ml and the dynamic range is 310 pg/ml. The preliminary results show the plasma α-synuclein for PD patients distributes from 6 to 30 fg/ml. For PDD patients, the concentration of plasma α-synuclein varies from 0.1 to 100 pg/ml. Whereas the concentration of plasma α-synuclein for healthy subjects is significantly lower than that of PD patients. The ultra-sensitive IMR by utilizing antibody-functionalized magnetic nanoparticles and high-Tc SQUID magnetometer is promising as a method to assay plasma α-synuclein, which is a potential biomarker for discriminating patients with PD or PDD.

U. S. Veterinary Immune Reagents Network: Progress with poultry immune reagents development

USDA-ARS?s Scientific Manuscript database

A major obstacle to advances in veterinary immunology and disease research is the lack of sufficient immunological reagents specific for veterinary animal species. In 2006, U. S. Veterinary Immune Reagent Network (VIRN) Consortium (www.vetimm.org) was developed to develop immune reagents against ma...

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.

Criteria for the selection of a solid phase to be used in immunoassays.

PubMed

Delagneau, J F; Masseyeff, R

1990-01-01

Heterogeneous immunoassays are very sensitive and only limited in terms of performance by non specific binding. They require separation of free from bound fractions and concomitant use of a solid phase coated with an immunoreactive component (i.e. immunosorbent). The improvement of these key immunosorbents is crucial and involves a great deal of expertise and capabilities. Specifications differ according to procedure (e.g. capture or competitive assay). Each routinely used solid phase, such as polystyrene wells, porous membrane or dispersible microbeads, presents specific performance characteristics, advantages, and drawbacks. Among the tasks to be implemented are optimization of the spatial orientation of immunological reagents, selection of the surface neutral hydrophilic support, acceleration of reactions by increasing the reactive surface area of the supports, streamlining and simplification of procedural steps. These various aspects are abundantly described and emphasized here.

Complex amine-based reagents

NASA Astrophysics Data System (ADS)

Suslov, S. Yu.; Kirilina, A. V.; Sergeev, I. A.; Zezyulya, T. V.; Sokolova, E. A.; Eremina, E. V.; Timofeev, N. V.

2017-03-01

Amines for a long time have been applied to maintaining water chemistry conditions (WCC) at power plants. However, making use of complex reagents that are the mixture of neutralizing and the filmforming amines, which may also contain other organic components, causes many disputes. This is mainly due to lack of reliable information about these components. The protective properties of any amine with regard to metal surfaces depend on several factors, which are considered in this article. The results of applying complex reagents to the protection of heating surfaces in industrial conditions and estimated behavior forecasts for various reagents under maintaining WCC on heat-recovery boilers with different thermal circuits are presented. The case of a two-drum heat-recovery boiler with in-line drums was used as an example, for which we present the calculated pH values for various brands of reagents under the same conditions. Work with different reagent brands and its analysis enabled us to derive a composition best suitable for the conditions of their practical applications in heat-recovery boilers at different pressures. Testing the new amine reagent performed at a CCPP power unit shows that this reagent is an adequate base for further development of reagents based on amine compounds. An example of testing a complex reagent is shown created with the participation of the authors within the framework the program of import substitution and its possible use is demonstrated for maintaining WCC of power-generating units of combined-cycle power plants (CCPP) and TPP. The compliance of the employed reagents with the standards of water chemistry conditions and protection of heating surfaces were assessed. The application of amine-containing reagents at power-generating units of TPP makes it possible to solve complex problems aimed at ensuring the sparing cleaning of heating surfaces from deposits and the implementation of conservation and management of water chemistry condition

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

PubMed

Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

2016-05-15

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

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

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

PubMed

Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

2018-01-01

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

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.

Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper.

PubMed

Barr, Katie; Korchagina, Elena; Ryzhov, Ivan; Bovin, Nicolai; Henry, Stephen

2014-10-01

Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Le(a) trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems. © 2014 AABB.

Paper-based microreactor integrating cell culture and subsequent immunoassay for the investigation of cellular phosphorylation.

PubMed

Lei, Kin Fong; Huang, Chia-Hao

2014-12-24

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum.

PubMed

Bryan, Donna; Silva, Nilupa; Rigsby, Peter; Dougall, Thomas; Corran, Patrick; Bowyer, Paul W; Ho, Mei Mei

2017-08-05

At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-1 19 , MSP-1 42 , MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross

Direct competitive chemiluminescence immunoassays based on gold-coated magnetic particles for detection of chloramphenicol.

PubMed

Liang, Xiaohui; Fang, Xiangyi; Yao, Manwen; Yang, Yucong; Li, Junfeng; Liu, Hongjun; Wang, Linyu

2016-02-01

Direct competitive chemiluminescence immunoassays (CLIA) based on gold-coated magnetic nanospheres (Au-MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au-MNPs were modified with carboxyl groups and amino groups by 11-mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). NSP-DMAE-NHS, a new and effective luminescence reagent, was employed to label anti-CAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a 'homemade' luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC50 ) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme-linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSP-DMAE-NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement. Copyright © 2015 John Wiley & Sons, Ltd.

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.

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.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

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

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.

2012-08-03

Sandwich immunoassay is the standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention. Albeit highly sensitive, the development of a specific immunoassay is rather time-consuming and associated with extremely high cost due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissue and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and great multiplexing capability. In this study, we report for the first time the developmentmore » of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed very good correlation (R2 values ranging from 0.90 to 0.99) in several independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, simultaneous measurement of free and total PSA and many other biomarkers can be performed in a single analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.« less

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

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

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.

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.

Reagent Target Request for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

NCI's Antibody Characterization Program provides reagents and other critical resources to support protein/peptide measurements and analysis. In an effort to produce and distribute well-characterized monoclonal antibodies to the scientific community, the program is seeking cancer related protein targets for antibody production and characterization for distribution to the research community. Submission Period: May 20, 2011 - July 1, 2011.

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.

Interfering lipoproteins in magnetic field-assisted agglutination of superparamagnetic particles immunoassay.

PubMed

Cauet, Gilles; Daynès, Aurélien; Temurok, Nevzat

2016-04-01

The technology of magnetic field-assisted immuno-agglutination of superparamagnetic particles allows sensitive detection of biomarkers in whole blood. However, we observed non-specific agglutination (NSA), due to interfering plasma proteins, that negatively affects C-reactive protein immunoassay. The objective of the study was to identify the plasma proteins involved and to eliminate these interferences. Plasma was fractionated by size exclusion HPLC and each fraction was tested for non-specific agglutination. In addition, plasma proteins bound to magnetic particles were analyzed by SDS-gel electrophoresis and identified by mass spectrometry. We found that NSA was due to the binding of some lipoproteins to the particles. NSA was observed in the presence of purified LDL and VLDL but not HDL. NSA was mediated by the binding of ApoB100 to magnetic particles through its heparin binding sites. These interferences could be eliminated by addition of heparin or other polyanions like dextran sulfate to the assay buffer. NSA results from the binding of some plasma lipoproteins to magnetic particles. The use of a polyanion to eliminate these interferences allows the formulation of a stable reagent.

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

Iodine(III) Reagents in Radical Chemistry

PubMed Central

2017-01-01

Conspectus The chemistry of hypervalent iodine(III) compounds has gained great interest over the past 30 years. Hypervalent iodine(III) compounds show valuable ionic reactivity due to their high electrophilicity but also express radical reactivity as single electron oxidants for carbon and heteroatom radical generation. Looking at ionic chemistry, these iodine(III) reagents can act as electrophiles to efficiently construct C–CF3, X–CF3 (X = heteroatom), C–Rf (Rf = perfluoroalkyl), X–Rf, C–N3, C–CN, S–CN, and C–X bonds. In some cases, a Lewis or a Bronsted acid is necessary to increase their electrophilicity. In these transformations, the iodine(III) compounds react as formal “CF3+”, “Rf+”, “N3+”, “Ar+”, “CN+”, and “X+” equivalents. On the other hand, one electron reduction of the I(III) reagents opens the door to the radical world, which is the topic of this Account that focuses on radical reactivity of hypervalent iodine(III) compounds such as the Togni reagent, Zhdankin reagent, diaryliodonium salts, aryliodonium ylides, aryl(cyano)iodonium triflates, and aryl(perfluoroalkyl)iodonium triflates. Radical generation starting with I(III) reagents can also occur via thermal or light mediated homolysis of the weak hypervalent bond in such reagents. This reactivity can be used for alkane C–H functionalization. We will address important pioneering work in the area but will mainly focus on studies that have been conducted by our group over the last 5 years. We entered the field by investigating transition metal free single electron reduction of Togni type reagents using the readily available sodium 2,2,6,6-tetramethylpiperidine-1-oxyl salt (TEMPONa) as an organic one electron reductant for clean generation of the trifluoromethyl radical and perfluoroalkyl radicals. That valuable approach was later successfully also applied to the generation of azidyl and aryl radicals starting with the corresponding benziodoxole (Zhdankin reagent

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.

Production of latex agglutination reagents for pneumococcal serotyping

PubMed Central

2013-01-01

Background The current ‘gold standard’ for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping. Results Here we describe a method for the production of latex agglutination reagents based on the passive adsorption of antibodies to latex particles. Sixty-five latex agglutination reagents were made using the PneuCarriage Project (PCP) method, of which 35 passed QC. The other 30 reagents failed QC due to auto-agglutination (n=2), no reactivity with target serotypes (n=8) or cross-reactivity with non-target serotypes (n=20). Dilution of antisera resulted in a further 27 reagents passing QC. The remaining three reagents passed QC when prepared without centrifugation and wash steps. Protein estimates indicated that latex reagents that failed QC when prepared using the PCP method passed when made with antiserum containing ≤ 500 μg/ml of protein. Sixty-one nasopharyngeal isolates were serotyped with our in-house latex agglutination reagents, with the results showing complete concordance with the Quellung reaction. Conclusions The method described here to produce latex agglutination reagents allows simple and efficient serotyping of pneumococci and may be applicable to latex agglutination reagents for typing or identification of other microorganisms. We recommend diluting antisera or removing centrifugation and wash steps for any latex reagents that fail QC. Our latex reagents are cost-effective, technically undemanding to prepare and remain stable for long periods of time, making them ideal for

Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Efficient Fabrication and Sealing of Chips Using a "Chip-Olate" Process.

PubMed

Temiz, Yuksel; Delamarche, Emmanuel

2017-01-01

The fabrication of silicon-based microfluidic chips is invaluable in supporting the development of many microfluidic concepts for research in the life sciences and in vitro diagnostic applications such as the realization of miniaturized immunoassays using capillary-driven chips. While being extremely abundant, the literature covering microfluidic chip fabrication and assay development might not have addressed properly the challenge of fabricating microfluidic chips on a wafer level or the need for dicing wafers to release chips that need then to be further processed, cleaned, rinsed, and dried one by one. Here, we describe the "chip-olate" process wherein microfluidic structures are formed on a silicon wafer, followed by partial dicing, cleaning, and drying steps. Then, integration of reagents (if any) can be done, followed by lamination of a sealing cover. Breaking by hand the partially diced wafer yields individual chips ready for use.

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...

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.

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.

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.

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 sensitive chemiluminescent immunoassay to detect Chromotrope FB (Chr FB) in foods.

PubMed

Xu, Kun; Long, Hao; Xing, Rongge; Yin, Yongmei; Eremin, Sergei A; Meng, Meng; Xi, Rimo

2017-03-01

Chromotrope FB (Chr FB) is a synthetic azo dye permitted for use in foods and medicines. An acceptable daily intake (ADI) of Chr FB was 0-0.5mg/kg in China. In this study, we synthesized a Chr FB hapten with an amino group to prepare its artificial immunogen. Polyclonal antibodies obtained from New Zealand rabbits were applied to develop an indirect competitive chemiluminescent immunoassay (icCLIA) to detect Chr FB in foods. A horseradish peroxidase (HRP)-luminol-H 2 O 2 system was used to yield CL signal with p-iodophenol as an enhancement reagent. The method showed good specificity towards Chr FB and could detect as low as 0.02ngmL -1 Chr FB in buffer, 0.07ngg -1 in yoghurt candy, 0.07ngg -1 in vitamin drink and 0.13ngg -1 in bread. Compared with HPLC method, the proposed method is more sensitive by two orders of magnitude. The accuracy and precision of this method are acceptable and comparable with HPLC method. Therefore, the proposed method could be used for rapid screening of Chr FB in the mentioned foodstuffs. Copyright © 2016. Published by Elsevier B.V.

Measurement of late-night salivary cortisol with an automated immunoassay system.

PubMed

Vogeser, Michael; Durner, Jürgen; Seliger, Ewald; Auernhammer, Christoph

2006-01-01

Measurement of late-night salivary cortisol concentrations is increasingly used as a screening test in suspected Cushing's syndrome. Cortisol concentrations are typically extremely low in late-night samples and discordant assay-specific reference ranges have been reported. Therefore, the aim of our study was to assess the analytical performance of the first automated cortisol immunoassay specified for salivary measurements and to establish late-night sampling reference-range data for this test. Salivary cortisol was measured using the Roche Cobas Cortisol assay (Roche Diagnostics). Five salivary pools in different concentration ranges were used to assess the inter-assay imprecision of this test in a two-centre evaluation protocol including two reagent lots. Linearity was tested by serial dilution. Salivary samples were obtained at 23:00 h from 100 apparently healthy volunteers using a commercially available salivary sampling device (Salivette, Sarstedt). A subset of 20 samples was used for method comparison with isotope dilution liquid chromatography-tandem mass spectrometry. Inter-assay coefficients of variation (n=20) between 11.6% and 40.4% were found for mean cortisol concentrations between 12.9 and 2.6 nmol/L, with an estimated functional sensitivity of approximately 5.0 nmol/L. The test also gave linear results in the lowest concentration range between 1.0 and 8.3 nmol/L. Mean late-night salivary cortisol of 5.0 nmol/L was found for healthy individuals; the absolute range was 1.4-16.7 nmol/L, and the 95th percentile was 8.9 nmol/L. Substantially lower concentrations were found with isotope dilution LC-MS/MS compared to immunoassay results (mean concentrations 1.8 and 4.4 nmol/L, respectively). The automated assay investigated was found to offer acceptable analytical performance in the very low concentration range required for late-night salivary cortisol, despite a very short turn-around time. Using this assay, late-night salivary cortisol concentrations

40 CFR 160.83 - Reagents and solutions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Reagents and solutions. 160.83 Section 160.83 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS GOOD LABORATORY PRACTICE STANDARDS Testing Facilities Operation § 160.83 Reagents and solutions. All reagents and...

Polyborylated reagents for modern organic synthesis

PubMed Central

SHIMIZU, Masaki; HIYAMA, Tamejiro

2008-01-01

Diverse kinds of gem- and vic-diborylated compounds are now readily available thanks to advances in gem-diborylation of lithium carbenoids as well as vic-diborylation of carbon–carbon multiple bonds with diboron compounds. These diborylated reagents lead to invention of polyborylated reagents and many novel and useful synthetic methods for supreme stereocontrol. This review summarizes preparative methods and synthetic reactions of di- and polyborylated reagents with the emphasis on multiple bond formation. PMID:18941288

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

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.

21 CFR 864.8100 - Bothrops atrox reagent.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

21 CFR 864.8100 - Bothrops atrox reagent.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

21 CFR 864.8100 - Bothrops atrox reagent.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Reagent Red Blood Cells. 660.30 Section 660.30 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be Reagent Red...

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.

Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3.

PubMed

Hong, Chenglin; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying

2009-02-09

The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 UmL(-1) with a coefficient 0.9986 and a detection limit of 0.64 UmL(-1) at S/N=3.

21 CFR 660.20 - Blood Grouping Reagent.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Blood Grouping Reagent. 660.20 Section 660.20 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.20 Blood Grouping Reagent. (a) Proper name and definition. The proper name of this product shall be Blood Grouping...

21 CFR 660.20 - Blood Grouping Reagent.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Blood Grouping Reagent. 660.20 Section 660.20 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.20 Blood Grouping Reagent. (a) Proper name and definition. The proper name of this product shall be Blood Grouping...

21 CFR 660.20 - Blood Grouping Reagent.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Blood Grouping Reagent. 660.20 Section 660.20 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.20 Blood Grouping Reagent. (a) Proper name and definition. The proper name of this product shall be Blood Grouping...

21 CFR 660.20 - Blood Grouping Reagent.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Blood Grouping Reagent. 660.20 Section 660.20 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.20 Blood Grouping Reagent. (a) Proper name and definition. The proper name of this product shall be Blood Grouping...

21 CFR 660.20 - Blood Grouping Reagent.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Blood Grouping Reagent. 660.20 Section 660.20 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.20 Blood Grouping Reagent. (a) Proper name and definition. The proper name of this product shall be Blood Grouping...

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.

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.

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.

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

Inactivation of rabies diagnostic reagents by gamma radiation

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

Gamble, W.C.; Chappell, W.A.; George, E.H.

1980-11-01

Treatment of CVS-11 rabies adsorbing suspensions and street rabies infected mouse brains with gamma radiation resulted in inactivated reagents that are safer to distribute and use. These irradiated reagents were as sensitive and reactive as the nonirradiated control reagents.

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

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

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

2000-01-01

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

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.

Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

NASA Astrophysics Data System (ADS)

Serebrennikova, Kseniya; Samsonova, Jeanne; Osipov, Alexander

2018-06-01

Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5-10 ng mL-1 with the limit of detection of 0.1 ng mL-1, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies).

Ranitidine interference with standard amphetamine immunoassay.

PubMed

Liu, Li; Wheeler, Sarah E; Rymer, Jacqueline A; Lower, Darla; Zona, Jayne; Peck Palmer, Octavia M; Tamama, Kenichi

2015-01-01

We recently encountered several cases of possible false positive results of amphetamine on the Beckman Coulter AMPH assay, but not on the Siemens EMIT II Plus assay. Our clinical chart review suggested that ranitidine interference may be responsible for the false positive results of the AMPH assays. Blank urine specimens spiked with ranitidine concentrations ranging from 5μg/ml to 5mg/ml were analyzed on both the AMPH and EMIT II Plus assays. To examine if the false positive results were due to assay specific reagent/sample ratios, we prepared 3 different sample to reagent ratios and analyzed them for amphetamine reaction rates on both assays. Ranitidine at 160μg/ml caused a positive interference on the AMPH assay. No interference was observed by ranitidine on the EMIT II Plus assay. Specifically, the sample to reagent ratios tested neither eliminated the positive inference on the AMP assay nor introduced an interference on the EMIT II Plus assay. Unlike the EMIT II Plus assay, the AMPH assay has cross-activity with ranitidine, which is independent of sample to reagent ratio. Copyright © 2014 Elsevier B.V. All rights reserved.

Modern affinity reagents: Recombinant antibodies and aptamers.

PubMed

Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

2015-12-01

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

TREATMENT OF MTBE USING FENTON'S REAGENT

EPA Science Inventory

This paper addresses the removal of MTBE from water, using Fenton's Reagent. Although complete mineralization of MTBE by Fenton's Reagent was not achieved, greater than 99% destruction of MTBE was realized. This was accomplished at a Fe+2:H2O2 ratio of 1:1 and one hour of contact...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

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.

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.

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.

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

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

PubMed

Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

2016-07-01

We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

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.

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.

Virus inactivation by nucleic acid extraction reagents.

PubMed

Blow, Jamie A; Dohm, David J; Negley, Diane L; Mores, Christopher N

2004-08-01

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

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.

Constructing New Bioorthogonal Reagents and Reactions.

PubMed

Row, R David; Prescher, Jennifer A

2018-05-15

Chemical tools are transforming our understanding of biomolecules and living systems. Included in this group are bioorthogonal reagents-functional groups that are inert to most biological species, but can be selectively ligated with complementary probes, even in live cells and whole organisms. Applications of these tools have revealed fundamental new insights into biomolecule structure and function-information often beyond the reach of genetic approaches. In many cases, the knowledge gained from bioorthogonal probes has enabled new questions to be asked and innovative research to be pursued. Thus, the continued development and application of these tools promises to both refine our view of biological systems and facilitate new discoveries. Despite decades of achievements in bioorthogonal chemistry, limitations remain. Several reagents are too large or insufficiently stable for use in cellular environments. Many bioorthogonal groups also cross-react with one another, restricting them to singular tasks. In this Account, we describe our work to address some of the voids in the bioorthogonal toolbox. Our efforts to date have focused on small reagents with a high degree of tunability: cyclopropenes, triazines, and cyclopropenones. These motifs react selectively with complementary reagents, and their unique features are enabling new pursuits in biology. The Account is organized by common themes that emerged in our development of novel bioorthogonal reagents and reactions. First, natural product structures can serve as valuable starting points for probe design. Cyclopropene, triazine, and cyclopropenone motifs are all found in natural products, suggesting that they would be metabolically stable and compatible with a variety of living systems. Second, fine-tuning bioorthogonal reagents is essential for their successful translation to biological systems. Different applications demand different types of probes; thus, generating a collection of tools that span a continuum of

A hybrid approach to urine drug testing using high-resolution mass spectrometry and select immunoassays.

PubMed

McMillin, Gwendolyn A; Marin, Stephanie J; Johnson-Davis, Kamisha L; Lawlor, Bryan G; Strathmann, Frederick G

2015-02-01

The major objective of this research was to propose a simplified approach for the evaluation of medication adherence in chronic pain management patients, using liquid chromatography time-of-flight (TOF) mass spectrometry, performed in parallel with select homogeneous enzyme immunoassays (HEIAs). We called it a "hybrid" approach to urine drug testing. The hybrid approach was defined based on anticipated positivity rates, availability of commercial reagents for HEIAs, and assay performance, particularly analytical sensitivity and specificity for drug(s) of interest. Subsequent to implementation of the hybrid approach, time to result was compared with that observed with other urine drug testing approaches. Opioids, benzodiazepines, zolpidem, amphetamine-like stimulants, and methylphenidate metabolite were detected by TOF mass spectrometry to maximize specificity and sensitivity of these 37 drug analytes. Barbiturates, cannabinoid metabolite, carisoprodol, cocaine metabolite, ethyl glucuronide, methadone, phencyclidine, propoxyphene, and tramadol were detected by HEIAs that performed adequately and/or for which positivity rates were very low. Time to result was significantly reduced compared with the traditional approach. The hybrid approach to urine drug testing provides a simplified and analytically specific testing process that minimizes the need for secondary confirmation. Copyright© by the American Society for Clinical Pathology.

21 CFR 866.3900 - Varicella-zoster virus serological reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Varicella-zoster virus serological reagents. 866... Varicella-zoster virus serological reagents. (a) Identification. Varicella-zoster virus serological reagents... viruses and provides epidemiological information on these diseases. Varicella (chicken pox) is a mild...

21 CFR 866.3900 - Varicella-zoster virus serological reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Varicella-zoster virus serological reagents. 866... Varicella-zoster virus serological reagents. (a) Identification. Varicella-zoster virus serological reagents... viruses and provides epidemiological information on these diseases. Varicella (chicken pox) is a mild...

21 CFR 866.3900 - Varicella-zoster virus serological reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Varicella-zoster virus serological reagents. 866... Varicella-zoster virus serological reagents. (a) Identification. Varicella-zoster virus serological reagents... viruses and provides epidemiological information on these diseases. Varicella (chicken pox) is a mild...

21 CFR 866.3900 - Varicella-zoster virus serological reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Varicella-zoster virus serological reagents. 866... Varicella-zoster virus serological reagents. (a) Identification. Varicella-zoster virus serological reagents... viruses and provides epidemiological information on these diseases. Varicella (chicken pox) is a mild...

21 CFR 866.3900 - Varicella-zoster virus serological reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Varicella-zoster virus serological reagents. 866... Varicella-zoster virus serological reagents. (a) Identification. Varicella-zoster virus serological reagents... viruses and provides epidemiological information on these diseases. Varicella (chicken pox) is a mild...

Chemical Amplification with Encapsulated Reagents

NASA Technical Reports Server (NTRS)

Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

2002-01-01

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

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.

40 CFR 792.83 - Reagents and solutions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 31 2010-07-01 2010-07-01 true Reagents and solutions. 792.83 Section 792.83 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) GOOD LABORATORY PRACTICE STANDARDS Testing Facilities Operation § 792.83 Reagents and...

21 CFR 866.3175 - Cytomegalovirus serological reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cytomegalovirus serological reagents. 866.3175 Section 866.3175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3175...

21 CFR 866.3145 - Coxsackievirus serological reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Coxsackievirus serological reagents. 866.3145 Section 866.3145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145...

21 CFR 866.3175 - Cytomegalovirus serological reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cytomegalovirus serological reagents. 866.3175 Section 866.3175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3175...

21 CFR 866.3145 - Coxsackievirus serological reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Coxsackievirus serological reagents. 866.3145 Section 866.3145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145...

21 CFR 866.3145 - Coxsackievirus serological reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Coxsackievirus serological reagents. 866.3145 Section 866.3145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145...

21 CFR 866.3145 - Coxsackievirus serological reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Coxsackievirus serological reagents. 866.3145 Section 866.3145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145...

21 CFR 866.3175 - Cytomegalovirus serological reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cytomegalovirus serological reagents. 866.3175 Section 866.3175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3175...

21 CFR 866.3145 - Coxsackievirus serological reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Coxsackievirus serological reagents. 866.3145 Section 866.3145 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145...

21 CFR 866.3175 - Cytomegalovirus serological reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cytomegalovirus serological reagents. 866.3175 Section 866.3175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3175...

21 CFR 58.83 - Reagents and solutions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL GOOD LABORATORY PRACTICE FOR NONCLINICAL LABORATORY STUDIES Testing Facilities Operation § 58.83 Reagents and solutions. All reagents and solutions in the laboratory areas shall be labeled to indicate identity, titer...

Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow.

PubMed

Taylor, Angela E; Keevil, Brian; Huhtaniemi, Ilpo T

2015-08-01

The recent onslaught of mass spectrometry (MS) to measurements of steroid hormones, including demands that they should be the only acceptable method, has confused clinicians and scientists who have relied for more than 40 years on a variety of immunoassay (IA) methods in steroid hormone measurements. There is little doubt that MS methods with their superior specificity will be the future method of choice in many clinical and research applications of steroid hormone measurement. However, the majority of steroid measurements are currently, and will continue to be, carried out using various types of IAs for several reasons, including their technical ease, cost and availability of commercial reagents. Speedy replacement of all IAs with MS is an unrealistic and unnecessary goal, because the availability of MS measurements is limited by cost, need of expensive equipment, technical demands and lack of commercial applications. Furthermore, IAs have multiple well-known advantages that vindicate their continuing use. The purpose of this article is to elucidate the advantages and limitations of the MS and IA techniques from two angles, i.e. promotion of MS and defence of IA. The purpose of the text is to give the reader an unbiased view about the current state and future trends of steroid analysis and to help him/her choose the correct assay method to serve his/her diagnostic and research needs. © 2015 European Society of Endocrinology.

21 CFR 866.3940 - West Nile virus serological reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false West Nile virus serological reagents. 866.3940... virus serological reagents. (a) Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum...

21 CFR 866.3940 - West Nile virus serological reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false West Nile virus serological reagents. 866.3940... virus serological reagents. (a) Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum...

21 CFR 866.3940 - West Nile virus serological reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false West Nile virus serological reagents. 866.3940... virus serological reagents. (a) Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum...

21 CFR 866.3940 - West Nile virus serological reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false West Nile virus serological reagents. 866.3940... virus serological reagents. (a) Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum...

21 CFR 866.3940 - West Nile virus serological reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false West Nile virus serological reagents. 866.3940... virus serological reagents. (a) Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum...

Strategies to prepare and use functionalized organometallic reagents.

PubMed

Klatt, Thomas; Markiewicz, John T; Sämann, Christoph; Knochel, Paul

2014-05-16

Polyfunctional zinc and magnesium organometallic reagents occupy a central position in organic synthesis. Most organic functional groups are tolerated by zinc organometallic reagents, and Csp(2)-centered magnesium organometallic reagents are compatible with important functional groups, such as the ester, aryl ketone, nitro, cyano, and amide functions. This excellent chemoselectivity gives zinc- and magnesium-organometallic reagents a central position in modern organic synthesis. Efficient and general preparations of these organometallic reagents, as well as their most practical and useful reactions, are presented in this Perspective. As starting materials, a broad range of organic halides (iodides, bromides, and also to some extent chlorides) can be used for the direct insertion of magnesium or zinc powder; the presence of LiCl very efficiently promotes such insertions. Alternatively, aromatic or heterocyclic bromides also undergo a smooth bromine-magnesium exchange when treated with i-PrMgCl·LiCl. Alternative precursors of zinc and magnesium reagents are polyfunctionalized aryl and heteroaryl molecules, which undergo directed metalations with sterically hindered TMP bases (TMP = 2,2,6,6-tetramethylpiperide) of magnesium and zinc. This powerful C-H functionalization method gives access to polyfunctional heterocyclic zinc and magnesium reagents, which undergo efficient reactions with numerous electrophiles. The compatibility of the strong TMP-bases with BF3·OEt2 (formation of frustrated Lewis pairs) dramatically increases the scope of these metalations, giving for example, a practical access to magnesiated pyridines and pyrazines, which can be used as convenient building blocks for the preparation of biologically active molecules.

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

EPA Science Inventory

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

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