Human Tamm-Horsfall protein (THP) was isolated by salt precipitation from pooled normal urine. Monoclonal antibodies were generated against this glycoprotein by fusion of human THP-primed mouse splenocytes with a mouse myeloma cell line. Six stable monoclonal antibody-producing clones were formed.
Each antibody was carefully characterized by three methods immunoperoxidase light microscopy, ELISA inhibition, and Western blotting. Once the specificity of the monoclonal antibodies had been established, they were used to study the distribution of THP in human renal tissue, and measure the concentration of the glycoprotein in serum.
One of these monoclonal antibodies (THPC4) was used to develop an immunogold technique to study the ultrastructural localization of THP in human kidneys. Results showed that THP had a discrete renal distribution, being associated with the luminal plasmalemma of distal convoluted tubules, and the whole cell plasmalemma of epithelium lining the thick ascending limb of Henle's loop. Despite some reports in the literature, THP appears to be mainly a membrane-associated protein. This may be related to its, as yet unknown, physiological role.
An ELISA assay, again using THPC4, was established to try and determine concentrations of THP in serum of normal subjects. A range of 80-620 ng/mL was found.
The use of monoclonal antibodies of defined specificity has provided the means for a more reliable assessment of levels of THP in serum, and distribution within the kidney. It may now be possible to determine a physiological role and pathological significance of THP in renal disease.[Show full abstract]
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