Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin: a study of sources of variation in assay results.
We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml-1, cm-1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.
Tracy
R P
RP
Department of Pathology, University of Vermont, Burlington 05405.
Andrianorivo
A
A
Riggs
B L
BL
Mann
K G
KG
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D005069:Evaluation Studies as Topic; D006461:Hemolysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015675:Osteocalcin / Q000097:blood* / Q000276:immunology / Q000592:standards; D011786:Quality Control; D012016:Reference Values
D000911:Antibodies, Monoclonal; D015675:Osteocalcin