BACKGROUND Although detection of natural haptens by antihapten antibodies in sandwich assay format has the theoretical advantages of high analytical specificity and sensitivity, this type of assay has not been reported because of the seemingly insurmountable task of avoiding steric hindrance between the 2 bindings. This is especially true for ring-structured hydrophobic haptens. The macrolide drug tacrolimus (FK506, Prograf®, 804 Da) is such a hapten. Here we show the detection of tacrolimus using 2 antitacrolimus monoclonal antibodies in a sandwich assay. METHODS Both antibodies were developed by use of an intact tacrolimus molecule covalently linked to a carrier protein but via 2 different positions separated by 10 carbon atoms. Epitope analysis based on drug analog binding was used to show no overlap between the binding sites of the 2 antibodies, indicating the 10-carbon separation resulted in 2 distinct epitopes. The distinct epitopes suggested that the drug might be approachable by the antibodies from 2 separate directions, which predicted simultaneous binding as in sandwich formation. RESULTS This prediction was confirmed in sandwich ELISA and affinity column–mediated immunoassay formats. The assay demonstrated good imprecision and significantly lower metabolite cross-reactivity than competitive assay counterparts. Comparison with liquid chromatography–tandem mass spectrometry (LC-MS/MS) using 55 whole-blood samples from transplant patients with tacrolimus concentrations ranging from 0.9 to 29.5 ng/mL showed a linear regression: sandwich = 0.99 × LC-MS/MS + 0.10 ng/mL, r = 0.991, Sy|x = 1.08 ng/mL. CONCLUSIONS This work demonstrates that a highly specific sandwich assay using 2 antihapten antibodies is feasible for the measurement of a hapten drug.
Recent clinical data indicate that the measurement of the concentration of C-reactive protein (CRP) requires a higher sensitivity and wider dynamic range than most of the current methods can offer. Our goal was to develop a totally automated and highly sensitive CRP assay with an extended range on the Dimension® clinical chemistry system based on particle-enhanced turbidimetric-immunoassay (PETIA) technology. The improved method was optimized and compared to the Binding Site's radial immunodiffusion assay using disease state specimens to minimize interference. Assay performance was assessed on the Dimension® system in a 12-instrument inter-laboratory comparison study. A split-sample comparison (n = 622) was performed between the improved CRP method on the Dimension® system and the N Latex CRP mono method on the Behring Nephelometer, using a number of reagent and calibrator lots on multiple instruments. The method was also referenced to the standard material, CRM470, provided by the International Federation of Clinical Chemistry (IFCC). The improved CRP method was linear to 265.1mg/l with a detection limit between 0.2 and 0.5mg/l. The method detects antigen excess from the upper assay limit to 2000mg/l, thereby allowing users to retest the sample with dilution. Calibration was stable for 60 days. The within-run reproducibility (CV) was less than 5.1% and total reproducibility ranged from 1.1 to 6.7% between 3.3 and 265.4mg/l CRP. Linear regression analysis of the results on the improved Dimension® method (DM) versus the Behring Nephelometer (BN) yielded the following equation: DM = 0.99 × BN − 0.37; r = 0.992. Minimal interference was observed from sera of patients with elevated IgM, IgG and IgA. The recovery of the IFCC standard was within 100 ± 7 % across multiple lots of reagent and calibrator. The improved CRP method provided a sensitive, accurate and rapid approach to quantify CRP in serum and plasma on the Dimension® clinical chemistry system. The ability to detect antigen excess eliminated reporting falsely low results caused by the ‘prozone effect’.
Background: Monitoring whole-blood concentrations of cyclosporin A (CsA) is common practice in the management of solid organ and bone marrow transplant recipients. In a multicenter study we evaluated a new, direct (no pretreatment) CsA assay on the Dade Behring Dimension RxLTM system and compared results with those from the Abbott TDx CsA immunoassay and a HPLC method. Methods: Whole-blood samples from heart (n = 111; 35 patients), liver (n = 201; 44 patients), kidney (n = 279; 65 patients), and miscellaneous organ (n = 77; 12 lung, 12 bone marrow, 5 kidney/pancreas, and 1 pancreas patient) recipients were obtained from patient populations of the participating institutions. Routine clinical monitoring of CsA was performed using either the TDx method or HPLC. Results: The minimum detectable concentration of CsA averaged 9.4 μg/L, and the lower limit of quantification was 30 μg/L. The method was linear from 30 to 500 μg/L. Cross-reactivity with seven different CsA metabolites ranged from 0.0% to 5.7% for the Dimension RxL assay compared with 0.4–15.9% for the TDx assay. Total imprecision (CV) averaged 6.2%, and within-run imprecision averaged 4.9%. Passing–Bablok linear regression analyses of all samples from two sites yielded the following: RxL = 0.81 × TDx − 16.8; and RxL = 1.12 × HPLC − 1.7. Conclusions: The Dade Behring CsA assay for the random-access Dimension platform offers adequate performance characteristics for routine clinical use, does not require a manual pretreatment step, and demonstrates less cross-reactivity with CsA metabolites than another commonly used immunoassay.
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