Abstract
Thrombotic thrombocytopenic purpura (TTP), a serious disorder characterized by the development of VWF-platelet rich thrombi in the arterioles and capillaries, requires reliable diagnostic assays for optimal management of the patients. Simple, reliable assays of ADAMTS13 inhibitors or antibodies are not yet widely available. To develop an ELISA for antibodies of ADAMTS13, we analyze the levels of patient IgG bound to immobilized recombinant ADAMTS13 in a microtiter plate format. The mean (±SD) IgG binding value is 5.1 ± 1.5 arbitrary units (AU)/mL among a group of 36 normal subjects and is 5.3 ± 2.7 AU/mL among 31 random patients (P > 0.1). Two (5%) of the normal group and 2 (6%) of the random group are outliers. When applied to patients with thrombotic microangiopathy, this assay yields IgG binding values ranging from 2.0 – 58.3 (median = 5.4) AU/mL in a group of 40 subjects with other thrombotic microangiopathy and 10.1 - > 60.0 (median 49.0) AU/mL in a group of 56 patients with acute TTP. Excluding 2 (5%) outlying samples, the mean (±SD) for the “other TM” group is 5.9 ±2.5 AU/mL, which is not different from the values of the normal and random subjects. In contrast, the IgG binding values are > 10.0 AU/mL in each of the acute TTP patients. Furthermore, heating the plasma samples at 56oC, presumably by dissociating ADAMTS13 from immune complexes, increases IgG binding of 4 weakly positive TTP samples from 10.1 – 12.7 to 28.3 – 154.3 AU/mL. Addition of native ADAMTS13 lacking the tag sequences decreases the binding of TTP IgG to 40% ± 20% of control, but does not affect the binding of 6 outlying non-TTP IgG (120% ± 20% vs. control, P < 0.001), suggesting that the IgG of non-TTP samples do not recognize ADAMTS13. In summary, the ELISA for ADAMTS13 antibodies is highly sensitive for detection of ADAMTS13 antibodies. A subset of the general population contains IgG molecules that react with the recombinant ADAMTS13 fusion protein but not with native ADAMTS13. Blocking with native ADAMTS13 enhances the specificity of the assay for TTP.
Left: Binding of IgG to immobilized rADAMTS13-FLAG/His in four groups of study subjects.
Right: Suppression of IgG binding by untagged ADAMTS13 in TTP but not in non-TTP samples.
Left: Binding of IgG to immobilized rADAMTS13-FLAG/His in four groups of study subjects.
Right: Suppression of IgG binding by untagged ADAMTS13 in TTP but not in non-TTP samples.
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