Immunoluminometric assay for the midregion of pro-atrial natriuretic peptide in human plasma.

The prohormone of atrial natriuretic peptide (proANP) is a polypeptide of 126 amino acids. Mature atrial natriuretic peptide (ANP) consists of amino acids 99–126 and comprises 98% of the natriuretic peptides in the circulation (1 ). The N-terminal portion of proANP, termed proANP1–98 or NT-proANP, has a much longer half-life than mature ANP and has therefore been suggested to be a more reliable analyte for measurement than mature ANP (2 ). In addition to other established indications, proANP has recently gained much interest as a potential new marker in the field of sepsis (3 ), emphasizing the need for a reliable assay for this molecule. All sandwich immunoassays developed for proANP to date use an antibody against the N-terminal region of proANP1–98 combined with a second antibody against either the midregion (4 ) or C-terminal region (5, 6). However, under certain conditions, the N-terminal region might be minimally accessible for antibody binding (7, 8). Despite the described long half-life of proANP, results from various competitive immunoassays as well as HPLC analyses indicate that proANP1–98 can be subject to further fragmentation (9, 10). We developed a new sandwich immunoassay for midregional proANP (amino acids 53–90; Fig. 1A). With this assay, which was named B.R.A.H.M.S SERISTRA (B.R.A.H.M.S AG), we measured proANP values in 325 healthy controls. EDTA-plasma samples were collected at a blood bank (Red Cross) from 325 consecutive healthy blood donors (age range, 18–67 years; 52.9% male) without clinical evidence of acute disease or a history of chronic illness. Blood donors with risk factors for heart failure were not enrolled in the study. Written consent was obtained from all donors. For the proANP assay, tubes were coated with affinitypurified polyclonal sheep antibodies specific for amino acids 73–90 (GRGPWDSSDRSALLKSKL) of the molecule (Fig. 1A). Coating of the antibody was done for 20 h on polystyrene tubes (1.5 g/tube) in 0.3 mL of buffer (10 mmol/L Tris-HCl, pH 7.8; 10 mmol/L NaCl). Tubes were blocked with 10 mmol/L sodium phosphate buffer containing 30 g/L of the polysaccharide Karion FP (Merck AG) and 5 g/L protease-free bovine serum albumin (Sigma), pH 6.8, and were lyophilized. A polyclonal sheep antibody specific for another part of proANP was used as tracer. This antibody was raised to peptide 53–72 (PEVPPWTGEVSPAQRDGGAL) of proANP and was affinity purified on a peptide-sulfolink column. After purification, the antibody was labeled with acridinium ester as follows: 100 g of antibody in 20 mmol/L sodium phosphate buffer, pH 8.0, was incubated for 20 min at room temperature with 10 L of acridinium ester (1 g/L in acetonitrile; Hoechst AG). Labeled antibody was purified by HPLC with a Knauer hydroxyapatite column (buffer gradient, 1–500 mmol/L potassium phosphate, pH 6.8; flow rate, 0.8 mL/min). Plasma proANP was measured as follows: 20 L of patient sample (EDTA plasma) or calibrator was added in duplicate to antibody-coated tubes containing 50 mmol/L sodium phosphate buffer, pH 7.5, and incubated for 2 h at room temperature. After five washes with 2 mL of standard LUMItest washing buffer (B.R.A.H.M.S AG), 200 L of tracer containing acridinium ester-labeled antiproANP antibody was added, followed by a 30-min incubation at room temperature. Tubes were washed three times with 2 mL of washing buffer, and detection was performed in a luminometer (detection time, 1 s/ sample; Berthold LB952T). The relative light units reported by the chemiluminescence assay were converted to pmol/L proANP as calculated from a calibration curve that was included in every analytical run. To prepare calibrators, proANP53–90, which contains Fig. 1. Representation of the assay principle for midregional proANP (A), and interassay imprecision for 25 plasma samples measured on 10 different days (B).