Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement.
Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.
Dressendörfer
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RA
Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
Kirschbaum
C
C
Rohde
W
W
Stahl
F
F
Strasburger
C J
CJ
D001710:Biotin / Q000378:metabolism*; D003429:Cross Reactions; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012469:Salivary Glands / Q000737:chemistry*
D001710:Biotin; D006854:Hydrocortisone