Generation, Characterization, and Use of Monoclonal Antibodies against Parathyroid Hypertensive Factor

[摘要] Parathyroid hypertensive factor (PHF) may be useful as a diagnostic marker of salt-sensitive, low-renin hypertension. PHF was discovered in the plasma of salt-sensitive hypertensive humans (1) and was also found to be increased in spontaneously hypertensive rats, DOCA-salt-hypertensive rats, and Dahl-salt-sensitive rats, but not in two-kidney one-clip rats or in Dahl-salt-insensitive rats (2)(3). Studies on the mechanism of PHF action indicate that this substance acts directly on vascular smooth muscle cells to enhance Ca2+ influx (4), likely associated with depolarization of the plasma membrane by inhibition of voltage-gated K+ channels (5). Together these actions will sensitize vascular tissues to other vasoconstrictors, such as norepinephrine and angiotensin II (6). PHF may therefore be a causative factor in the development of hypertension in some individuals. It was found that PHF-positive (salt-sensitive) patients respond best to calcium channel blockers and diuretics and that PHF-negative patients respond better to angiotensin-converting enzyme inhibitors and beta blockers (7). Recently, an enzyme immunoassay for detection of PHF in human plasma has been reported that uses anti-PHF oligoclonal antibodies (8). The present study describes the further development, characterization, and clinical application of monoclonal antibodies (MAbs) against PHF.Male BALB/c mice were immunized with partially purified PHF (9) prepared from medium harvested from cultured parathyroid glands of spontaneously hypertensive rats, as was described previously (10). Splenocytes were harvested and fused with SP/0-2 myeloma cells by standard procedures (11). The recloning procedure involved limiting-dilution conditions in 96-well plates (0.3 cells/well).The bovine serum albumin (BSA) conjugate of PHF was synthesized by standard procedures (12) and used in a direct ELISA for anti-PHF antibodies. Microtiter plates were coated with BSA-PHF (100 μL/well; 1:2000 dilution). After the plates were washed with phosphate-buffered saline (PBS) containing 5 mL/L Tween 20, supernatants of hybridoma cell cultures were added (100 μL/well) and …

[学科分类] 过敏症与临床免疫学