Affinity MALDI-MS of IgE

Since IgE normally occurs at levels of 800 pM in human serum (Gould et al., 

its detection in 10 - to lO -fold dilutions of serum is consistent with the detection of 1 pM to 100 fM IgE in standard IgE solutions. This detectability compares favorably with other published results, including label-free (Liss et al., 2002 Xu et al., 2005) and fluorescent-labeled (Gokulrangan et al., 2005  

Stadtherr et al., 2005) detection of IgE in simple solution that provided detectability down to 100 pM IgE [corresponding to 5 fmol using a 50-tiL aliquot in the case of one immobilized aptamer sensor (Xu et al., 2005)], and use of fluorescent-labeled IgE aptamer in the run buffer in affinity CE that gave a detection limit of 46 pM IgE in simple solution but yielded detectable signals only for serum that was spiked with 5 nM IgE and not for native IgE in the serum (German et al., 1998). In the present work we achieved capture and detection of native IgE in human serum and found that dilution of the serum by at least 10 -fold allowed detection of native IgE with little interference from other serum proteins. This is comparable to detectability of a commercial antibody-based ELISA kit that offers 75 pM detection (Stadtherr et al., 2005). 

The capacity of a typical aptamer-coated spot that is 1 cm in diameter is approximately 10 protein molecules. If 1 p,L of serum contains approximately 10 total protein molecules, dilution of the sample by 10 reduces the total protein molecules in the l-p,L application volume to 10, which is much less than the binding capacity of the spot. Dilution of the sample should therefore increase the availability of aptamer sites to IgE and accelerate equilibration. 

The studies described in this section demonstrate the power and simplicity of affinity MALDl-MS using aptamer-modified surfaces to capture and detect low-abundance proteins in biological fluids, to preconcentrate proteins directly at the MALDl probe surface, and to confirm the identity of the affinity-captured protein through examination of the mass spectrum. The reusable surfaces could easily be adapted to microarray formats, and the procedure is amenable to automation. 

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