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Clin Biochem. 2013 Aug;46(12):963-968. doi: 10.1016/j.clinbiochem.2013.01.012. Epub 2013 Jan 28.

A comparison of capture antibody fragments in cardiac troponin I immunoassay.

Author information

1
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland. Electronic address: hmhyyt@utu.fi.
2
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland.

Abstract

OBJECTIVES:

To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody.

DESIGN AND METHODS:

The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab')2 fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24-60 years).

RESULTS:

The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P<0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab')2) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab.

CONCLUSIONS:

Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption.

KEYWORDS:

Antibody fragment; Cardiac troponin I; Heterophilic antibody; Human anti-mouse antibodies; Immunoassay; Nanoparticle

[Indexed for MEDLINE]

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