Two-site time-resolved immunofluorometric assay of human insulin.

Toivonen, E.; Hemmilä, Ilkka; Marniemi, Jukka; Jørgensen, P. N.; Zeuthen, Jesper; Lövgren, Timo

doi:10.1093/clinchem/32.4.637

Cited by 61 publications

(17 citation statements)

References 9 publications

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“…d. From LOD to upper calibrator. and 2 sandwich immunofluorometric assays, 16,17 which were all based the on the 2 antibodies HUI018 and OXI005. The working range was similar for LOCI™ and the immunofluorometric assays, whereas it was smaller for the ELISA.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

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

Poulsen¹,

Jensen²

2007

SLAS Discovery

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“…However, we are not aware of any assays of insulin which have been characterized in detail with regard to their crossreactivity with the proinsulin-like molecules and which in particular have been shown to give operational specificity for insulin. When cross-reactivity with proinsulin-like molecules has been assessed it has been studied using intact proinsulin (Yoshioka et al, 1979;Ruan et al, 1986;Tolvonen et al, 1986;Comitti et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins

Sobey

Beer

Carrington

et al. 1989

Biochemical Journal

342

204

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Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.

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“…Serum free fatty acids (FFAs) were measured using an enzymatic colorimetric procedure with a ‘NEFA C’ kit (Wako Chemicals GmbH, Neuss, Germany). Serum insulin and C‐peptide were measured by a noncompetitive time‐resolved immunofluorometric assay (TR‐IFMA) (Wallac Oy, Turku, Finland), as described previously [22,23].…”

Section: Methodsmentioning

confidence: 99%

Effect of insulin on protein phosphatase 2A expression in muscle in type 2 diabetes

Højlund

Poulsen

Stæhr

et al. 2002

Eur J Clin Investigation

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Two-site time-resolved immunofluorometric assay of human insulin.

Cited by 61 publications

References 9 publications

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

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

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins

Effect of insulin on protein phosphatase 2A expression in muscle in type 2 diabetes

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