Radioimmunoassay for Rat B-Type Natriuretic Peptide (BNP-45)

Abstract

Rat BNP-45 is the main circulating form of BNP in rat plasma. To understand the role of BNP in physiological and pathophysiological conditions, a specific radioimmunoassay (RIA) for the quantitative determination of the peptide in plasma and tissues is necessary. An assay using rBNP-45 as the standard in conjunction with antisera directed against this peptide has not been described in the literature, though some investigators have reported values ranging from 0.73-2.0 pmol/L using either BNP-26 or BNP-32 as the standard peptide. Unfortunately, these forms of BNP do not exist in rat plasma. In our studies, we have developed a specific RIA for rBNP-45 using rBNP-45 as the standard peptide and Tyro-rBNP-45 as the radioligand. We have used two specific antisera for assay purposes; one against rBNP-45, and the second to a peptide composed of the first 20 amino acids of rBNP-45 (rBNP[1-20]). The recovery of various amounts of rBNP-45 added to control plasma was 50-80% depending on the method of extraction and purification. The interassay and intraassay coefficients of variation were 12% and 6% respectively. Values obtained were similar for blood sampled by either cardiac puncture, decapitation, or aortic puncture. The method was used to measure rBNP-45 in the plasma of normal (WKY) and Spontaneously Hypertensive (SHR) rats. The values obtained were 5.46 +/- 0.43 and 19.6 +/- 2.36 pmol/L respectively. The rat atrial natriuretic peptide (ANP[99-126]) values in the same extracts were 23.2 +/- 0.45 and 51.6 +/- 3.16 pmol/L.