A "Mix and Read" Assay for Insulin Using Fluorometric Microvolume Assay Technology

Toney, Jeffrey H.; Ogawa, Aimie M.; Blair, Megan; Park, Young-Whan

doi:10.1089/154065803322302772

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…Two technologies to note are the homogenous fluorometric microvolume assay technology (FMAT) [35] and ECL [36]. FMAT can be formatted such that biotinylated antigen interacts with streptavidin coated beads.…”

Section: Alphalisamentioning

confidence: 99%

The Use of AlphaScreen Technology in HTS: Current Status

Eglen¹,

Reisine²,

Roby³

et al. 2008

Curr Chem Genomics

221

184

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AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

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Section: Alphalisamentioning

confidence: 99%

The Use of AlphaScreen Technology in HTS: Current Status

Eglen¹,

Reisine²,

Roby³

et al. 2008

Curr Chem Genomics

221

184

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“…Homogenous insulin sandwich immunoassays, such as FMAT 5 and HTRF, 6 have been described for serum and culture medium measurements. Heterogeneous assays with a built-in automated wash procedure, such as electrochemiluminescence (ECL), 7 the Gyrolab Bioaffi™ system, 8 and the Luminex system, 9 with many of the advantages of homogenous assays, have also been developed.…”

Section: Introductionmentioning

confidence: 99%

“…Comparison of the LOCI™ assay with some homogenous or homogenous-like insulin assays such as ECL (rat insulin in serum), 7 HTRF (rat insulin in medium), 6 Gyrolab (insulin-Asp in human serum), 8 Luminex (human insulin in human plasma), 9 and FMAT (rat insulin in rat plasma) 5 is more difficult because the antibodies, insulin, and sample types are different. Overall, the LOCI™ seems superior when considering a palette of important performance characteristics, such as sensitivity, analytical range, sample volume, assay time, and ease of handling.…”

mentioning

confidence: 99%

A Luminescent Oxygen Channeling Immunoassay for the Determination of Insulin in Human Plasma

Poulsen¹,

Jensen²

2007

SLAS Discovery

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The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.

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“…These vary from assays within a single liquid phase followed by turbidimetric, 4 colorimetric, [5][6][7] fluorometric, 8,9 and luminescent 10 detection schemes to biphasic assays in which reagents partition between a liquid and a solid phase. Examples of the latter include beads, [11][12][13][14] colloidal gold and latex particles, 15,16 membranes, 17,18 and cells. [19][20][21] Some authors have argued that the term homogeneous be restricted to assays where the solid phase remains suspended in solution, 22 however, there is no general agreement on this point.…”

Section: Introductionmentioning

confidence: 99%

Confocal detection of planar homogeneous and heterogeneous immunosorbent assays

et al. 2009

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Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

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A "Mix and Read" Assay for Insulin Using Fluorometric Microvolume Assay Technology

Cited by 6 publications

References 9 publications

The Use of AlphaScreen Technology in HTS: Current Status

The Use of AlphaScreen Technology in HTS: Current Status

A Luminescent Oxygen Channeling Immunoassay for the Determination of Insulin in Human Plasma

Confocal detection of planar homogeneous and heterogeneous immunosorbent assays

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