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

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.

Rapid and sensitive cardiac troponin I immunoassay based on fluorescent europium(III)-chelate-dyed nanoparticles.

BACKGROUND: Cardiac troponins are the preferred and recommended biomarkers of myocardial infarction. Unfortunately, most of the current commercial assays do not meet the guideline recommendations for sensitivity and low-end precision. Therefore, improvements in their analytical performance are still needed. METHODS: Cardiac troponin I (cTnI) immunoassay was developed. The assay utilized a monoclonal antibody and a F(ab')(2) antibody fragment immobilized onto the microtiter wells for capturing, and a monoclonal antibody covalently conjugated to fluorescent europium(III)-chelate-dyed nanoparticles for detecting. Following a 15-min incubation of the sample and nanoparticle-bioconjugates in the capture wells, cTnI was quantified directly from the washed well surface by time-resolved fluorometry. RESULTS: The limits of detection and quantification were 0.0020 µg/l and 0.012 µg/l, respectively. The response was linear in the measured range of 0.003-9.6 µg/l. The within-run imprecisions were 9.8, 5.1, 7.7 and 5.4%, and the total imprecisions were 13.1, 10.4, 9.0 and 8.7% at cTnI levels of 0.007, 0.051, 0.52 and 2.62 µg/l, respectively. Plasma recoveries of added cTnI were 72-119%. Regression analysis with Innotrac Aio! 2nd generation cTnI assay yielded a slope (95% confidence intervals) of 1.197 (1.141 to 1.253) and y-intercept of 0.216 (-0.128 to 0.561)µg/l (S(yx) = 2.176 µg/l, n = 212, r = 0.945). CONCLUSIONS: The developed immunoassay based on europium(III)-chelate-dyed nanoparticle label allows rapid and sensitive measurement of cTnI.

Upconverting phosphors in a dual-parameter LRET-based hybridization assay.

Upconverting phosphors (UCPs) are lanthanide-doped sub-micrometer-sized particles, which produce multiple narrow and well-separated anti-Stokes emission bands at visible wavelengths under infrared excitation (980 nm). The advantageous features of UCPs were utilized to construct a dual-parameter, homogeneous sandwich hybridization assay based on a UCP donor and lanthanide resonance energy transfer (LRET). UCPs with two emission bands (540 nm and 653 nm) were exploited together with two appropriate fluorophores as acceptors. The energy transfer excited emissions of the acceptors were measured at 600 nm and 740 nm without any significant interference from each other. The autofluorescence limitation associated with conventional fluorescence was totally avoided as the measurements were carried out at shorter wavelength relative to the excitation. In the sandwich hybridization assay two different single-stranded target-oligonucleotide sequences were detected simultaneously and quantitatively with a dynamic range from 0.03 to 0.4 pmol (corresponding 0.35-5.4 nM). The UCPs enable multiplexed homogeneous LRET-based assay requiring only a single excitation wavelength, which simplifies the detection and extends the applicability of upconversion in bioanalytical measurements.