A time-resolved fluoroimmunoassay for cortisol in unextracted bovine plasma or serum with optimized procedures to eliminate steroid binding protein interference and to minimize non-specific streptavidin-europium binding.
A time-resolved fluoroimmunoassay (TR-FIA) for human salivary cortisol was adapted for the measurement of cortisol in unextracted bovine blood plasma and serum. It has been demonstrated that the binding of cortisol binding plasma proteins (CBPP) to the cortisol-biotin primary probe cannot be eliminated by means of cortisol releasing agents. Complete inactivation of CBPP was achieved by heating water diluted samples for 30 min at 80 degrees C. The high non-specific binding (NSB) of the streptavidin-europium secondary probe, encountered during preliminary experiments, was shown to be caused by an interaction with bovine serum albumin (BSA) and could be reduced partly by the addition of heparin. It was also shown that the ability to bind streptavidin-europium nonspecifically is not a general property of proteins since bovine gamma-globulin and gelatin lack this behaviour. The advantage of a highly reduced NSB, resulting from the use of a BSA free assay buffer, is not limited to this particular assay but is also beneficial for other procedures based on specific measurement of streptavidin-europium fluorescence. The detection limit for a 20 microl sample was 0.5 ng/ml. The intra-assay coefficients of variation for control samples with cortisol concentrations of 71.1, 39.2 and 10.3 ng/ml were 8.2, 7.9 and 11.3% (n = 16). The corresponding inter-assay coefficients of variation were 7.3, 9.0 and 11.2% (n = 73). Correlation with a commercially available radioimmunoassay, preceded by diethylether extraction of the sample, was 0.97 (n = 88).
Erkens
J H
JH
Department of Reproduction, DLO-Institute for Animal Science and Health (ID-DLO), Lelystad, The Netherlands.
Dieleman
S J
SJ
Dressendörfer
R A
RA
Strasburger
C J
CJ
D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D005063:Europium; D005453:Fluorescence; D006801:Humans; D006854:Hydrocortisone / Q000097:blood*; D007118:Immunoassay; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013997:Time Factors
D002352:Carrier Proteins; C027091:cortisol binding globulin; D005063:Europium; D019809:Streptavidin; D006854:Hydrocortisone