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


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.
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland. Electronic address: hmhyyt@utu.fi.
Heidi
H
Hyytiä
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland.
Marja-Leena
ML
Järvenpää
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland.
Noora
N
Ristiniemi
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland.
Timo
T
Lövgren
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland.
Kim
K
Pettersson
D000328:Adult; D002138:Calibration; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000097:blood / Q000378:metabolism*; D007700:Kinetics; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D019210:Troponin I / Q000097:blood / Q000378:metabolism*; D055815:Young Adult
D007128:Immunoglobulin Fragments; D019210:Troponin I
Antibody fragment; Cardiac troponin I; Heterophilic antibody; Human anti-mouse antibodies; Immunoassay; Nanoparticle