Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

Ishikawa, Eiji; Ishikawa, Setsuko; Hashida, Seiichi; Hashinaka, Kazuya

doi:10.1002/(sici)1098-2825(1998)12:3<154::aid-jcla5>3.0.co;2-9

Cited by 5 publications

(2 citation statements)

References 18 publications

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“…That is, our method also has the potential to reach limits of detection of 10 −20 mol/assay. Ishikawa and Hashida 30 , 31 have achieved the limit of detection of 10 −20 mol/assay for HIV-1 p24 by their own method, the ‘immune complex transfer enzyme immunoassay’. This method, however, requires time and effort for the detection compared with our method.…”

Section: Methodsmentioning

confidence: 99%

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Watabe¹,

Kodama

Kaneda

et al. 2014

BIOPHYSICS

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An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

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Section: Methodsmentioning

confidence: 99%

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Watabe¹,

Kodama

Kaneda

et al. 2014

BIOPHYSICS

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show abstract

“…For example, an immune complex transfer enzyme immunoassay showed a limit of detection of 0.24 pg/mL (i.e., ca . 1 x 10 −17 moles/mL) [ 6 , 7 ]. A cytometric bead assay was developed to detect p24 at 0.4 pg/mL (i.e., ca .…”

Section: Introductionmentioning

confidence: 99%

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

et al. 2015

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To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

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Designing allosteric peptide ligands targeting a globular protein

et al. 2006

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Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein‐targeted, L or D retro‐inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G‐protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, β‐galactosidase, βGAL. Three of the eight 14mer peptides allosterically activated βGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 × 10−9 by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their βGAL binding‐mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the βGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy–entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein‐targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 38–59, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

Cited by 5 publications

References 18 publications

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

Designing allosteric peptide ligands targeting a globular protein

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