Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques
Claudia Alpini1, Milvia Lotzniker2, Serenella Valaperta1
1Servizio Analisi Chimico Cliniche Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
Insights
Characterizing anti-cytoplasmic antibodies using advanced techniques like confocal microscopy and Western blot is crucial. These methods help identify specific autoantibodies, moving them from "esoteric" to "diagnostic" markers.
Area of Science:
- Immunology
- Cell Biology
Background:
- Anti-cytoplasmic antibodies are complex and not fully characterized by standard indirect immunofluorescence (IIF).
- Home-made morphological and biochemical techniques are explored for better characterization.
Purpose of the Study:
- To characterize anti-cytoplasmic antibodies using advanced morphological and biochemical methods.
- To overcome limitations of indirect immunofluorescence (IIF) in identifying complex cytoplasmic antigens.
Main Methods:
- Tested nine serum samples with anti-cytoplasmic antibodies using fluorescent confocal microscopy, Western blot, and electron microscopy.
- Utilized specific antibody markers for confirmation and identified targets like calreticulin and LC3B.
Main Results:
- Confocal microscopy confirmed IIF staining patterns.
- Electron microscopy validated anti-endoplasmic reticulum positivity.
- Western blot definitively identified anti-lysosome/endosome and anti-endoplasmic reticulum antibodies via calreticulin and LC3B reactivity.
Conclusions:
- Confocal microscopy can be utilized in reference laboratories.
- Electron microscopy is not suitable for routine diagnostics.
- Advanced techniques like multiplex immunoassay offer easier autoantibody identification, aiding in defining clinical relevance.
Purpose:
The aim of our study was the characterization of anti-cytoplasmic antibodies by home-made morphological and biochemical techniques. Indeed, indirect immunofluorescence (IIF) on HEp-2 cell line is not always exhaustive in relation to the complexity of the antigens involved.
Methods:
Nine serum samples with anti-cytoplasmic antibodies (2 anti-Golgi apparatus, 3 with diffuse pattern and 4 with lysosome/endosome-like pattern) were tested with fluorescent confocal microscopy, Western blot analysis and, when necessary, with electron microscopy technique.
Results:
Confirmation of the IIF staining pattern was performed in confocal microscopy by comparison with the respective antibody marker. The anti-endoplasmatic reticulum positivity was also confirmed by electron microscopy evaluation. Both anti-lysosome/endosome and anti-endoplasmatic reticulum positivity have been definitely identified by Western blot through clear reactivity with calreticulin and LC3B, respectively.
Conclusions:
These results do not aim at representing a standard routine laboratory procedure. Electron microscopy evaluation cannot be proposed as a routine approach, but confocal microscopy technique may be offered in centralized reference laboratories. Newer technologies, especially multiplex immunoassay, can also lead to an easier identification of these autoantibodies, without recurring to a home-made immunoblotting. Only with a complete characterization we will be able to define the clinical relevance of anti-cytoplasmic antibodies, which are still considered as "esoteric" and not as "diagnostic" antibodies.
