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.

Bloodstream infection is an important cause of death among leukemia patients, and the etiologic agent surveillance is important for the prophylaxis and treatment. This study aims to identify the common bloodstream isolates in hospitalized leukemia patients with septicemia in our hospital, to choose the ideal combination of antimicrobial agents for infection prophylaxis and to clarify the appropriate time for antibiotic prophylaxis. To know this information, a retrospective analysis was conducted over a 9-year period from July 2001 to July 2010 by reviewing medical records of leukemia children admitted to our hospital. The overall frequencies of isolation were 45% in Gram-positive bacteria, 53.8% in Gram-negative bacteria, and 1.2% in fungi, respectively. Coagulase-negative Staphylococci were the most common organisms isolated, accounting for 32.7% of the total blood culture isolates, followed by Escherichia coli (15.7%) and Klebsiella pneumoniae (7.1%). The incidence of septicemia caused by extended-spectrum β-lactamase-producing E. coli and K. pneumoniae was high (69.2% and 58.8% of total isolates, respectively). The coverage rate of antimicrobial combinations of "vancomycin+cefoperazone-sulbactam" and "vancomycin+piperacillin-tazobactam" to blood culture isolates of leukemia patients in our hospital were 91.88% and 90.27%, respectively. More than 90% of septicemia occurred when the absolute neutrophil count was lower than 1.6×10⁹/L and 83.05% when absolute neutrophil count was lower than 1.0×10⁹/L. These results suggest that ongoing surveillance for antimicrobial susceptibility in leukemia children remains essential. Vancomycin+cefoperazone-sulbactam and vancomycin+piperacillin-tazobactam are the good choice for leukemia children to prevent bacterial infections in our hospital. In an effort to reduce total consumption of antibiotics and to elevate the therapeutic efficacy, antibiotics prophylaxis should be started with the appearance of neutropenia in leukemia children.

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