Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique.

PURPOSE: Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or immunoprecipitation plus mass spectrometry. Thus, sera are commonly pre-screened for interesting immunopatterns via immunocytochemistry/immunohistochemistry. However, distinguishing immunopatterns can be difficult and intracellular antigens are less accessible. Therefore, we present an alternative: a simple and cheap immunoblot screening able to detect proteins refractory to solubilization.

EXPERIMENTAL DESIGN: We revised five steps of immunoblotting-based autoantigen screening: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, and (5) antigen detection. Thereafter, we screened the sera of 52 patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls.

RESULTS: The protein source strongly impacts the set of detected antigens. Protein extraction and protein separation can be adapted for refractory proteins. Further, antigen detection should be improved by applying a ≥75% time- and material-saving longitudinal cutting of protein lanes. This allows for exact comparison of band patterns. As these are individually highly specific and temporarily constant, we refer to them as "immunological fingerprints". In a proof-of-principle, we detected a 155 kDa immunoband with two anti-neurofascin-155 (NF-155)-positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3 to 6 out of 52 CIDP patients.

CONCLUSION AND CLINICAL RELEVANCE: Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens. This article is protected by copyright. All rights reserved.