N008: Detection of parathyroid hypertensive factor in parathyroid cell culture media

Parathyroid hypertensive factor (PHF) is a circulating hypertensive factor found in a proportion of human essential hypertensive patients and spontaneously hypertensive rats. This study describes a new immunoassay system for detection of PHF in rat parathyroid cell culture media. We have developed a hybridoma which secreted specific anti-PHF antibodies of IgM isotype. Research study demonstrated no detectable cross-reactivity with rat parathyroid hormone or shorter parathyroid hormone fragments, different vasoactive substances and plasma proteins. PHF assay is a one step competitive assay using a microtiter plates coated with monoclonal antibodies. Culture media supernatant is diluted (1:10) in a specimen diluent and incubated with horseradish peroxidase labeled PHF in microtiter plate wells. The assay has a dynamic range of 0–2.0 units/ml, with lower detection limit of 0.02 units/ml. Assay reproducibility was determined by testing three samples over the dynamic range; a precision of 14% (inter assay) and 8.3% (intra assay) was achieved. PHF immunoassay results are in agreement with previously used bioassay and show significant difference between the level of PHF secreted by parathyroid cells from spontaneously hypertensive and Wistar-Kyoto rats. In conclusion PHF enzyme immunoassay is rapid, highly sensitive and can provide a useful tool for further investigation of the physiological role of PHF.