To the Editor
We thank Corrado Murtas et al. for their attention to our paper that demonstrated the importance of anti-α enolase (AENO) antibodies in primary and secondary membranous nephropathy (MN). We emphasized the presence of anti-AENO IgG1 and IgG4 subclasses of antibodies in primary MN, and IgG1 and IgG3 subclasses in secondary MN, including lupus nephritis (type V) and bucillamine-related MN [1].
Murtas et al. reported the coexistence of different circulating anti-podocyte antibodies in MN [2]. They emphasized anti-AENO IgG4 antibodies. In the present study, our data were consistent with theirs for primary MN. However, for secondary MN, depending on autoimmune type, IgG1 and IgG3 subclasses were predominant. They also commented a high prevalence of anti-AENO IgG2 antibodies in lupus nephritis [3]. However, we did not detect anti-AENO IgG2 antibodies. We compared the dot blot methods for anti-AENO antibody between our study and Bruschi’s study (Table 1). Major differences are blocking reagents for nonspecific binding (skim milk versus BSA), and the dilution titers of patients’ sera (1:200 versus 1:50) and antibody for human IgG subclasses (1:500 versus 1:3000). Further study will be needed to confirm the best method for detecting anti-AENO antibody.
Table 1.
A comparison on dot blot methods for anti-α enolase (AENO) antibody
| Kimura et al. [1] | Bruschi et al. [3] | |
|---|---|---|
| Patients | 25 patients with primary MN, and 13 patients with lupus nephritis (V) | 20 patients with lupus nephritis (II, III, IV, V) |
| Nitrocellulose membranes | Nitrocellulose membranes (GE Healthcare, UK) | Bio-Dot apparatus (Bio-Rad, Hercules, CA, USA) |
| Antigen: α-enolase | Recombinant: home made | Recombinant: Abnova Corporation (Taipei, Taiwan) |
| Antigen amounts | 10 μg/ml of enolase in Tween-PBS, for 60 min at room temperature | 100 ng of enolase in TBS, for 4 h at room temperature, and then at 4 °C overnight |
| Blocking of nonspecific binding | 5 % (w/v) skim milk (BD Difco, USA) | 5 % (w/v) albumin in Tween-PBS |
| Patients’ sera | Diluted 1:200 in Tween-PBS at room temperature overnight | Diluted 1:50 in TBS-T (TBS-T 0.05 % v/v, 1 % w/v albumin) for 6 h at room temperature, and then at 4 °C overnight |
| Antibody | Peroxidase–conjugated mouse monoclonal antibodies to human IgG1, IgG2, IgG3, IgG4 (Invitrogen, USA) at diluted 1:500 for 2 h at room temperature | HPR–anti human IgG1, IgG2, IgG3, IgG4 (Invitrogen, USA) at diluted 1:3000 for 4 h at room temperature |
| Results | 18 of 25 sera (IgG1 and IgG4, 11 patients; IgG4 alone, 6 patients; IgG1 alone, 1 patient) showed anti-AENO antibody in primary MN, and positive IgG1 and IgG3 anti-AENO antibody in 9 out of 13 patients with lupus nephritis (V) | Strong positive IgG2 anti-AENO antibody in lupus nephritis (II, III, IV, V) |
Regard no detection of α-enolase in glomeruli from primary MN in our study, we used human IgG-adsorbed primary and secondary antibodies tested on frozen sections gave no signal of α-enolase in glomeruli but still well visible staining in proximal tubules. These discrepancies between the two studies should be investigated further in a future study.
We agree with Dr. Murtas’s comments that the combination of antigenic α-enolase epitopes and IgG subclasses may differ by disease. We have to pay attention to anti-AENO IgG subclasses, which may shed new light on the mechanism of MN and other diseases.
Compliance with ethical standards
Conflict of interest
All the authors have declared no competing interest.
Footnotes
This reply refers to the article available at doi:10.1007/s10157-016-1263-y.
References
- 1.Kimura Y, Miura N, Debiec H, Morita H, Yamada H, Banno S, Ronco P, Imai H. Circulating antibodies to alpha-enolase and phospholipase A2 receptor and composition of glomerular deposits in Japanese patients with primary or secondary membranous nephropathy. Clin Exp Nephrol. 2016 doi: 10.1007/s10157-016-1235-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Murtas C, Bruschi M, Candiano G, Moroni G, Magistroni R, Magnano A, Bruno F, Radice A, Furci L, Argentiero L, Carnevali ML, Messa P, Scolari F, Sinico RA, Gesualdo L, Fervenza FC, Allegri L, Ravani P, Ghiggeri GM. Coexistence of different circulating anti-podocyte antibodies in membranous nephropathy. Clin J Am Soc Nephrol. 2012;7(9):1394–1400. doi: 10.2215/CJN.02170312. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bruschi M, Sinico RA, Moroni G, Pratesi F, Migliorini P, Galetti M, Murtas C, Tincani A, Madaio M, Radice A, Franceschini F, Trezzi B, Bianchi L, Giallongo A, Gatti R, Tardanico R, Scaloni A, D’Ambrosio C, Carnevali ML, Messa P, Ravani P, Barbano G, Bianco B, Bonanni A, Scolari F, Martini A, Candiano G, Allegri L, Ghiggeri GM. Glomerular autoimmune multicomponents of human lupus nephritis in vivo: alpha-enolase and annexin AI. J Am Soc Nephrol. 2014;25(11):2483–2498. doi: 10.1681/ASN.2013090987. [DOI] [PMC free article] [PubMed] [Google Scholar]