Two-site immunofluorometric assay of intact salmon calcitonin with improved sensitivity.
We recently developed a two-site immunofluorometric assay (IFMA) of salmon calcitonin (SCT) by DELFIA (dissociation enhancement lanthanide fluoroimmunoassay) technique using the same polyclonal antibodies both for "catching" the antigen and for signaling. In the present study we used a monoclonal antibody to SCT 1-11 as the capture antibody. This antibody was biotinylated before use in streptavidin-coated microtitration plates. The polyclonal antibody labeled with Eu chelate was used as a signaling marker. This combination of antibodies resulted in an assay that was three to four times more sensitive than the previous IFMA, with a detection limit of 0.3 pmol/L serum. Intact SCT 1-32 was detected by the assay (recoveries 94-96%), but not the fragments SCT 1-11 and SCT 10-32 or human calcitonin. Dilutions of plasma samples containing SCT were parallel to the calibration curve of SCT 1-32. Pharmacokinetic studies of SCT, 100 IU administered intramuscularly to 10 men, indicated peak serum concentrations of 32-128 pmol/L within 10-20 min with apparent half-life of 56+/-18 min (mean+/-SD). This new assay will allow study of the pharmacokinetics of new calcitonin preparations that do not require injection.
Rong
H
H
Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Hospital and Institute, Stockholm, Sweden.
Deftos
L J
LJ
Ji
H
H
Bucht
E
E
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D001710:Biotin; D002116:Calcitonin / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D008297:Male; D008674:Metals, Rare Earth; D008875:Middle Aged; D010446:Peptide Fragments / Q000032:analysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D008674:Metals, Rare Earth; D010446:Peptide Fragments; D005063:Europium; D001710:Biotin; C028815:salmon calcitonin; D002116:Calcitonin