IgE-binding analysis by fluid-phase assay We performed a second set of experiments by using a reverse enzyme allergo-sorbent test that steps the binding of labeled allergens by individuals IgE antibodies captured by an anti-human IgE monoclonal antibody immobilized within the sound phase.30 Here, plates were first coated with an anti-human IgE monoclonal antibody (clone LE27). variable level of IgE cross-reactivity was exposed among the individuals. Nimodipine The mean portion of cross-reactive IgE antibodies displayed only 17.1% of 2S-albumins-specific IgE antibodies and was lower than the mean fraction of IgE specific to Ara h 2 (57.4%) or to Ara h 6 (25.5%). The higher level of Ara h 2-specific IgE was principally due to the IgE-binding capacity of an insertion comprising the repeated immunodominant linear epitope DPYSPOHS. The effect of IgE cross-reactivity on diagnostic screening was illustrated having a serum showing an Ara h 6-specific IgE response of 26 UI/mL that was not associated with the capacity of Ara h 6 to result in mast cell degranulation. Conclusions & Clinical Relevance: IgE antibodies specific to peanut 2S-albumins are primarily non-cross-reactive but low-affinity cross-reactivity can affect diagnostic accuracy. Screening IgE binding to a mixture of 2S-albumins rather than to each separately may enhance diagnostic overall performance. manifestation plasmid pET9c (Novagen-Merck, Damstadt, Germany).22,28 The variant rAra h 2. was acquired by replacing the website GRDPYSPSQDPYSPSP of rAra h 2.01 Nimodipine by the dipeptide DS occurring naturally in Ara h 6. Expression, purification and refolding of recombinant proteins were performed as previously explained.22,29 Correct refolding of the recombinant proteins was verified by circular dichroism spectroscopy.22 The peptide containing three hydroxyprolines (pep 3POH, DPYSPOHSQDPYSPOHSQDPDRRDPYSPOHSPY) corresponds to the major linear epitope found in Ara h 2.02 isoform. The peptide was synthesized using a standard solid phase synthesis from the Fmoc (9-fluorenyl-methoxycarbonyl) continuous-flow method (peptide synthesizer 433A, Applied Biosystems, Foster City, CA). After standard process including TFA cleavage and ether precipitation, crude peptides were purified by RP-HPLC and characterized by MALDI-TOF.22 2.3. IgE-binding measurement by solid-phase assay IgE levels to individual native 2S-albumins were quantified using a direct enzyme allergosorbent test in which purified antigens (2.5 g/mL) were passively adsorbed on microtiter plates as previously described (Fig. 1A; Ara h 2, reddish triangle; Ara h 6, blue triangle).25 After overnight incubation with 2 or 3 3 dilutions of sera (50 L/well, Fig. 1A, step 1 1), IgE binding was exposed by the addition of a labeled anti-human IgE monoclonal antibody (clone BS17). Tracer was prepared by covalent linkage of the monoclonal antibody to the tetrameric form of acetylcholinesterase.25 After washing, Ellmans reagent was used as the enzyme substrate and absorbance was measured at 414 nm. Open in a separate window Number 1. IgE cross-reactivity between Ara h 2 and Ara h 6 investigated having a solid-phase assay. A, schematic basic principle of the solid-phase assay (Ara Nimodipine h 2, reddish triangle; Ara h 6, blue triangle, observe Methods); B, relative decrease of IgE binding to one 2S-albumin after depletion of IgE antibodies realizing the additional 2S-albumin (n=32, mean is definitely indicated). Cross-reactivity between Ara h 2 and Ara h 6 was evaluated by measuring the residual IgE binding to one 2S-albumin after depletion of IgE antibodies realizing the additional 2S-albumin. After GNG4 over night incubation of 50 L of diluted sera in microtiter plate Nimodipine coated with one 2S-albumin (Fig. 1A, step 1 1), 45 L of the producing depleted serum was transferred into another microtiter plate (Fig. 1A, step 2 2) coated either with the same 2S-albumin (in order to measure the effectiveness of IgE depletion) or with the additional 2S-albumin (in order to evaluate the decrease of IgE binding to that 2S-albumin, Fig. 1A, step 3 3). Depletion effectiveness of Ara h 2-specific IgE antibodies was 96.5 6.2% and depletion effectiveness of Ara h 6-specific IgE antibodies was 98.3 3.8% (n=32, data not shown). IgE levels to both 2S-albumins.
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