Development and evaluation of a practical ELISA for human urinary lipocalin-type prostaglandin D synthase.
BACKGROUND
Urinary excretion of lipocalin-type prostaglandin D synthase (L-PGDS) is significantly increased in patients with chronic renal failure, but its diagnostic potential in less advanced stages of renal diseases remains to be elucidated.


METHODS
Six mouse monoclonal antibodies (MAbs) were raised against recombinant human L-PGDS. We constructed a sandwich ELISA with two MAbs that recognized different epitopes with high affinities and assessed its assay performance and clinical utility with urine samples from healthy controls, diabetic patients, and patients with various renal diseases.


RESULTS
Western blot analyses with NH(2)-terminus-truncated L-PGDS mapped the epitopes to Ala(23)-Val(28) (MAb-7F5 and -10A3), Ser(52)-Ala(73) (MAb-9A6), Tyr(107)-Val(120) (MAb-1B7 and -6F5), and Gly(140)-Pro(155) (MAb-6B9). A sandwich ELISA was constructed with MAb-1B7 and -7F5, the K(d) values of which were 3.6 and 3.9 nmol/L, respectively, for native L-PGDS. Recoveries were 91-111%, and intra- and interassay CVs were <6% and <9%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by a variety of urinary constituents. Urinary L-PGDS excretion was significantly increased in patients with diabetic nephropathy, IgA nephropathy, and chronic glomerulonephritis even when serum creatinine was not increased. In patients with renal diseases, urinary L-PGDS was correlated with urinary albumin (r = 0.64; P <0.0001), N-acetyl-beta-D-glucosaminidase (r = 0.43; P <0.001), and serum creatinine (r = 0.66; P <0.0001). At a cutoff value of 284 mg/mol creatinine, the assay had sensitivities of 74% for diabetic nephropathy and 83% for chronic glomerulonephritis and a specificity of 93%.


CONCLUSIONS
This ELISA system is suitable for measurement of urinary L-PGDS in a routine clinical assay and may be useful to detect less advanced stages of renal diseases.
Oda
Hiroshi
H
Central Research Institute, Maruha Corp., 16-2, Wadai, Tsukuba, Ibaraki 300-4295, Japan.
Shiina
Yasuhiko
Y
Seiki
Kousuke
K
Sato
Nobuyuki
N
Eguchi
Naomi
N
Urade
Yoshihiro
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000652:urine; D002908:Chronic Disease; D003928:Diabetic Nephropathies / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005921:Glomerulonephritis / Q000276:immunology / Q000652:urine; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000276:immunology / Q000652:urine*; D054834:Lipocalins; D008297:Male; D051379:Mice; D008958:Models, Molecular
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D007070:Immunoglobulin A; D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase