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Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique
Author(s) -
Moritz Christian P.,
Tholance Yannick,
Rosier Carole,
ReynaudFederspiel Evelyne,
Svahn Juliette,
Camdessanché JeanPhilippe,
Antoine JeanChristophe
Publication year - 2019
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.201800157
Subject(s) - autoantibody , antigen , serology , chronic inflammatory demyelinating polyneuropathy , protein microarray , immunology , antibody , medicine , biology , microarray , biochemistry , gene , gene expression
Purpose Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or IP+MS. 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, a simple and cheap immunoblot screening able to distinguish immunopatterns and to detect refractory proteins is presented. Experimental Design Five steps of immunoblotting‐based autoantigen screening are revised: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, (5) antigen detection. Thereafter, 52 patients' sera with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls were screened. Results The protein source impacts the detected antigen set. Steps 2‐4 can be adapted for refractory proteins. Furthermore, longitudinal cutting of protein lanes saves ≥75% of time and material and allows for exact comparison of band patterns. As the latter are individually specific and temporarily constant, we call them “immunological fingerprints”. In a proof‐of‐principle, a 155 kDa immunoband was detected with two anti‐neurofascin‐155‐positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3‐6 of 52 CIDP patients. Conclusions and Clinical Relevance Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens.