CLIN.CHEM. 38/5, 628-635 (1992) 628 CLINICALCHEMISTRY, Vol. 38, No. 5, 1992 Multisite Immunochemiluminometric Assay for Simultaneously Measuring Whole-Molecule and Amino-Terminal Fragments of Human Parathyrin George G. Klee, Carol M. Preissner, Patricia G. Schryver, Robert L. Taylor, and Pal C. Kao The immunochemiluminometric assay described uses immobilized anti-human parathynn (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44--68) antisera and acridi- nium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal frag- ments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immu- noradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above- normal preoperative PTH concentrations, whereas pa- tients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of pn- mary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonethe- less, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive ami- no-terminal fragments will react in the assay if they are present in the patients’ sera or if they are produced by in vitro proteolysis of intact PTH. AddItional K.yphrases: adenoma hypercalcemia of mafig- nancy . cancer The measurement of human parathyrin (human para- thyroid hormone, hPTH) in peripheral blood is beset with several difficulties related to the heterogeneity of circulatory forms and the limited availability of specific high-affinity antisera. P’FH is synthesized in the para- thyroid glands and secreted mainly as an 84-amino-acid peptide that is degraded in the liver and other sites by cleavage in the 34-37 amino acid region (1). Normally, the amino-terminal fragments are cleared rapidly, whereas the carboxyl-termunal and mid-molecule frag- ments remain in circulation longer. The circulatory half-life of carboxyl-terminal fragments depends on re- nal function because they are cleared mainly by glomer- ular filtration. The net effect of these production and clearance rates is that the circulating concentration of carboxyl-t.erminal and mid-molecule fragments is greater than the concentration of intact molecule, espe- cially in patients with compromised renal function. Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905. Presented in part at the 41st National Meeting of the AACC, Atlanta, Georgia, July 23-27, 1989 (Chin Chem 1989;35:1147, abstract). Received July 29, 1991; accepted February 20, 1992. However, the functional activity of the P’FH molecule is related to the amino-terminal portion of the molecule. Assays for monitoring the biological activity of PTH should reliably measure both intact and functional amino-terminal fragments without interference from carboxyl- and mid-molecule fragments or other sub- stances. Our functional bioassay (bio-PTH) provides such a monitor by using anti-amino-terminal antisera to immunoextract and concentrate both hPTH(1-84) and the amino-terminal fragments, followed by mea- surement of their combined activity in stimulating the production of cAMP in cultured osteosarcoma cells (2). Our goals in developing the current assay were to have an assay that would correlate well with this bio-P’FH assay and also to have an assay that would be easier to perform and would measure lower concentrations of PTH. (A major problem in the development of reliable assays is the low peripheral blood concentration of intact and amino-terminal PrH.) The functional bioas- say achieved a measurement sensitivity of 1.0 pmol/L by extracting and concentrating 2.0 mL of plasma; how- ever, this is not sensitive enough to measure subnormal concentrations of the hormone. Therefore, we wanted to develop an assay capable of measuring P’FH concentra- tions well below the range found in normal subjects. Key factors in immunoassay sensitivity are the affin- ity of the antisera, the configuration of the assay, and the specific activity of the detection signal. Brown et al. (3) reported that immunoafflnity-purifled polyclonal an- tisera were more sensitive than monoclonal antisera for measuring P’FH. Miles and Hales (4) found that immu- nometric systems based on a combination of immobi- lized and labeled antibodies were more sensitive than competitive radioimmunoassays with labeled antigens. Ehrlich and Moyle (5) showed that combinations of antisera with affinities for multiple sites on an antigen can effectively increase the binding affinity. In addition, Weeks et al. (6) showed that acridinium esters can produce mmunoassay labels with high specific activi- ties. We have combinea each of these observations to develop a highly sensitive immunochemiluminometric assay (IcMA) that uses a combination of three affinity- purified polyclonal antisera. We evaluated the assay analytically and clinically by comparison with our func- tional bioassay, by comparison with a commercial im- munoradiometric assay, and by measuring PTH in plasma from normal subjects and patients with disor- ders of PTH metabolism. MaterIals and Methods Antisera Production Three polyclonal anti-hFFH antisera were used in this assay: two attached to the solid-phase capture