Comparison of two automated adrenocorticotropic hormone assays.

The measurement of adrenocorticotropic hormone (ACTH 1-39; Mr 4500) plays a key role in the evaluation of hyperand hypocortisolism (1–3), but low ACTH concentrations make accurate measurement challenging. Two automated, nonisotopic ACTH assays have become available in recent years. Because it is usual for rather small series to be assayed for ACTH in a clinical setting and because nonisotopic methods with stable calibration are of great practical interest, we compared these assays. Both assays investigated were solid-phase, sandwich immunoassays that use chemiluminescence for signal generation and are implemented on benchtop, multichannel, random-access analyzers with ready-to-use reagents. The Nichols Advantage ACTH assay (Nichols Institute Diagnostics) uses one acridinium ester-labeled mouse monoclonal antibody that specifically binds to the Cterminal region of ACTH and one biotin-labeled goat polyclonal antibody that binds to the N-terminal region. The sample is incubated for 21 min at 37 °C with both antibodies simultaneously, leading to the formation of a soluble sandwich complex in the presence of ACTH molecules. Streptavidin-coated magnetic particles are then added, and the reaction mixture is incubated for 10 min, during which the sandwich complex binds to the particles by biotin-avidin interaction. The particles are fixed to the wall of the single-use reaction cell magnetically, and unbound, labeled antibody is separated by aspiration and subsequent washing. Finally, two trigger solutions are injected into the reaction cells to initiate the chemiluminescent reaction, and the light emission is quantified over 2 s by a luminometer. A lot-specific master calibration curve is loaded by barcode and adjusted weekly with two calibrators by the user. The reported measuring range extends to 1500 ng/L. Barcodelabeled sample tubes are processed directly. The sample throughput is ;90/h, and the time to first result is 37 min with a start-up time of ;15 min. In the DPC IMMULITE ACTH assay (Diagnostic Products Corporation), a polystyrene bead within a test unit serves as the solid phase; the bead is coated with a monoclonal murine anti-ACTH antibody. The patient sample and a buffer are introduced and incubated for 30 min at 37 °C with intermittent agitation of the test unit. Unbound sample is then removed by a centrifugal wash procedure. Subsequently, an alkaline phosphatase-labeled polyclonal rabbit anti-ACTH antibody is introduced for a second 30-min cycle, after which unbound enzyme conjugate is removed by another centrifugal wash. Finally, the chemiluminescent substrate, a phosphate ester of adamantyl dioxetane, is added. This compound undergoes hydrolysis in the presence of alkaline phosphatase to yield an unstable intermediate, producing a sustained emission of light that is photomultiplied and quantified for 12 s. Adjustment of a predefined, lot-specific master calibration curve is performed with two calibrators at recommended intervals of 2 weeks. The reported measuring range is 10–1250 ng/L. The system reads sample barcodes after an aliquot of the sample has been pipetted into an assay cup and the test units have been placed with the cups on a load chain. The sample throughput is ;90 determinations per hour; the time to first result is 70 min with a start-up time of 10 min. Both assays were handled according to the manufacturers’ instructions in all respects by experienced technicians in a routine laboratory setting. Quality control was performed for all analytical runs using lyophilized control materials provided by the respective manufacturers (two concentrations for the DPC assay and three for the Nichols assay) according to the German Medical Association guidelines. The study protocol was approved by the institutional review board. Linearity of the assays was investigated by serial dilution of a high-ACTH patient sample (.1250 ng/L in the DPC assay and 1110 ng/L in the Nichols assay) with a low-ACTH pool in nine steps up to a dilution of 1:512. In both assays, results were linear (r .0.99). To study the interassay imprecision of the assays, four plasma pools were prepared from residual patient samples, aliquoted after equilibration, and then stored at 270 °C. ACTH was measured in 12 analytical runs using two different lots of reagents and five calibration cycles in both assays over a 3-month period. After thawing at 4 °C, the samples were centrifuged and split for analysis by both assays simultaneously; each sample was analyzed once in each assay. The results of the imprecision study are given in Table 1. For the method comparison study, 444 clinical EDTAplasma samples were used. In 42 of the samples, ACTH values determined with the DPC and Nichols assays were ,10 ng/L; in 17 samples, ACTH was .10 ng/L with the DPC assay and ,10 ng/L with the Nichols assay; in 37 samples, ACTH was ,10 ng/L with the DPC assay and .10 ng/L with the Nichols assay. Results for the remaining 348 samples were compared by unbiased regression analysis according to Deming: Nichols 5 0.81(DPC) 1 13.3 ng/L; 95% confidence interval for slope, 0.79–0.82; 95% confidence interval for intercept, 11.9–14.6 ng/L (Pearson r 5 0.982).