Underestimation of serum 25-hydroxyvitamin D by the Nichols Advantage Assay in patients receiving vitamin D replacement therapy.

was added to the well, and the lumi-nescence was measured (ALOKA lu-minometer). The IgG fraction of serum samples from the patient and from five control individuals was purified with a MAbTrap TM Kit (Amer-sham Biosciences), and the IgG concentration was adjusted to 4.0 g/L. The purified IgG (0.08 mL) was incu-bated at 4 °C for 24 h with 0.08 mL of PBS alone or with T 2 , T 3 , or T 4 (Sig-ma; at 4570, 42, and 768 nmol/L, respectively) dissolved in PBS. Each sample was mixed vigorously with 1.2 mL of polyethylene glycol (PEG; 125 g/L), centrifuged at 2800g for 30 min, aspirated, and washed with 1.2 mL of PEG (125 g/L). After the precipitates were dissolved in 0.001 mol/L hydrochloric acid (0.04 mL) and neutralized by equal amounts of 0.001 mol/L sodium hydroxide, T 2 and T 3 were measured by the FT 3 assay and T 4 by the FT 4 assay. The concentrations of T 2 were expressed as T 3 concentrations. Each sample was analyzed in duplicate. The ability of the purified IgG to bind T 2 , T 3 , or T 4 was defined as the difference between the FT 3 or FT 4 assay result and the respective blank value and is reported as the ␦T 2 , ␦T 3 , or ␦T 4 value. The ratios of FT 3 and FT 4 concentrations in PEG-treated samples (6) to those in untreated samples were significantly lower for the patient than for 37 other patients (Table 1 in the online Data Supplement); therefore , immunoglobulins in the pa-tient's serum interfered with both the FT 3 and FT 4 assays. FT 3 and FT 4 values in the mixtures of serum with sheep IgG, bovine globulin, and gel-atin did not differ significantly from those in the mixtures of serum and PBS only, suggesting that hetero-philic antibodies and anti-gelatin an-tibodies did not cause the high FT 3 and FT 4 values. When we examined the patient's IgG binding with Vitros FT3II and FT 4 assay wells, the luminescence generated by the patient's serum was higher than that of the 5 control individuals (Table 1 in the online Data Supplement). This suggested that the patient's IgG bound to T 2-and T 3-gelatin. The FT 3 and FT 4 concentrations in purified IgG and in treated samples of purified IgG (6) were below the lower …