Background Isotypes C4A and C4B of human complement component C4 are innate immunity factors. We found that gel electrophoresis of patient sera in the new diagnostic area of C4 complexes/aggregates (pI 4-5) followed by immunoblotting allowed evaluation of patient C4 isotype deficiency grouping in accordance to our developed functional C4 isotype analyses in microplates . Solid phase peroxidase kinetic chemiluminescence (CL) in the presence of CL substrate is sensitive process to detect serum glycoproteins distribution.
Objectives The objective was further study of patient C4B and C4A subisotypes within C4-isotype-(non-deficient or deficient) sera groups of patients.
Methods C4 subisotype patterns were obtained using separation of desialylated sera by isoelectric focusing in plate of polyacrylamide in gradient pH 3-5 followed by gel blotting on Immobillon P (Millipore). Blots were treated with goat anti-C4 polyclonal antibodies conjugated to horse radish peroxidase (Ab-HRP) or Ab-HRP against human IgG, IgM or IgA. Protein-A–HRP was also used. Substrate (BioWest, UVP; with increased sensitivity) was added and kinetics of the blot HRP CL was stepwise registered in live imagination regime using BioChemi System (UVP). Subisotypes were revealed by blot treatment with acetate buffer pH 4.5. Analysis was especially effective in optimized by us conditions (desialylation control, blot washings, substrate using, Cl registration regimes).
Results Patient C4B or C4A subisotypes were presented as combinations of mainly up to 5 bands of different contribution. Contribution of subisotype(s) in C4A region was confirmed by the presence of increased levels of IgG in less acidic band(s) and/or IgM as additional early CL of mostly acidic 5th band in cases of SLE with or without antiphospholipid syndrome (group of C4B-deficiency). In cases of non-C4-deficiency groups (examples of rheumatoid arthritis etc.) preferential localization of non-early IgM, IgA and binding Protein A within C4B region indicated infectious nature of contribution to subisotype(s) bands. Ig Fc-fragments were masked in complexes (C1q binding was not observed). Relative resistance of subisotypes patterns upon sera storing indicated partial masking glycans in C4 subisotypes complexes possessing increased hydrophobicity. Algorithm of revealing very low levels of serum subisotypes (invisible in comparison to other sera) is proposed.
Conclusions Results support both biomarker diagnostic and prognostic potential of patient sera C4B and C4A subisotypes and prospects of its further development of fine resolution CL patterns of C4 associates together with increased levels of classes and subclasses of Ig and lipoproteins, glyco- and lipid-containing antigens (also of microbial origin), others sera components using extended set of Ab. Few diagnostic kinetic CL pattern states (early and later) are possible to characterize disease(s) type.
Lakhtin M et al. Lakhtin M et al. Chemiluminescent visualization and quantification of the complement glycoprotein C4 containing systems in patient whole sera separated with isoelectric focusing in polyacrylamide gel and electroblotting: sialidase- and peroxidase-based new approaches. Glycoconjugate J 2011; 28: 296.
Disclosure of Interest None declared