ELISA for urinary trehalase with monoclonal antibodies: a technique for assessment of renal tubular damage.
BACKGROUND
alpha,alpha-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses alpha,alpha-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples.


METHODS
We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared.


RESULTS
The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7-7.6% and 6.2-8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities.


CONCLUSIONS
We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.
Ishihara
R
R
Department of Pediatrics, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8506, Japan. ishihara@takii.kmu.ac.jp
Taketani
S
S
Sasai-Takedatsu
M
M
Adachi
Y
Y
Kino
M
M
Furuya
A
A
Hanai
N
N
Tokunaga
R
R
Kobayashi
Y
Y
D000118:Acetylglucosaminidase / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D007231:Infant, Newborn; D007674:Kidney Diseases / Q000201:enzymology / Q000652:urine*; D007684:Kidney Tubules / Q000201:enzymology*; D008297:Male; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D014198:Trehalase / Q000276:immunology / Q000652:urine*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014198:Trehalase; D000118:Acetylglucosaminidase