Establishment of a sandwich ELISA for human megsin, a kidney-specific serine protease inhibitor.
BACKGROUND
We previously identified a novel serine protease inhibitor (serpin), megsin, which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion, suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases.


METHODS
We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases.


RESULTS
Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml, which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18), 55.6% were urinary megsin-positive, while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive, respectively. Among patients with chronic renal failure due to unknown causes (n = 74), 18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073), 12.3% were urinary megsin-positive, while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note, when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate, urinary megsin excretion increased as the stage progressed up to stage 3A, suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage, probably reflecting development of glomerulosclerosis.


CONCLUSIONS
We established a high-sensitive megsin ELISA, which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases.
Inagi
Reiko
R
Institute of Medical Sciences, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Izuhara
Yuko
Y
Tominaga
Naoto
N
Nangaku
Masaomi
M
Kurokawa
Kiyoshi
K
Miyata
Toshio
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005919:Glomerular Filtration Rate; D005921:Glomerulonephritis / Q000652:urine*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D012016:Reference Values; D012680:Sensitivity and Specificity; D015842:Serine Proteinase Inhibitors / Q000652:urine*; D015843:Serpins / Q000032:analysis / Q000737:chemistry / Q000652:urine*
D000911:Antibodies, Monoclonal; C114170:SERPINB7 protein, human; D015842:Serine Proteinase Inhibitors; C491938:Serpinb7 protein, mouse; C491937:Serpinb7 protein, rat; D015843:Serpins