Immunoluminometric Assay for the Midregion of Pro-Atrial Natriuretic Peptide in Human Plasma

Abstract

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; 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 (PEVP-PWTGEVSPAQRDGGAL) 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 t...

Breed Differences in Natriuretic Peptides in Healthy Dogs

“…Most breeds had higher concentrations of proANP 31‐67 than NT‐proBNP, which is consistent with recent findings in healthy people 27, 48. The interquartile distance was large in several breeds, especially for NT‐proBNP, with 32% of the total number of dogs having NT‐proBNP concentrations higher than a previously proposed value indicative of heart disease in dogs 30.…”

“…A high individual weekly variability in NT‐proBNP concentration has been shown in healthy dogs,32 making interpretation of a single individual sample uncertain. The lower variability for proANP 31‐67 in combination with high biologic stability, attributable to low risk of fragmentation,27 makes it an interesting molecule for further studies in dogs. Comparing concentrations of proANP 31‐67 in this study to the previously proposed cutoff value of 1,750 pmol/L for CHF in dogs,49 <1% of our included healthy dogs were above the cutoff.…”

“…Sample handling was standardized, although minor differences in short‐term freezing temperature occurred at 1 center. However, NPs have been shown to be stable at the freezing temperatures used,27, 36, 37 and each peptide was analyzed in batches at the same laboratory. Hence, it is unlikely that sample handling had any major effect on the results.…”

“…For practical reasons, at 1 center samples were stored at −20°C for a maximum of 2 weeks, after which they were transferred frozen to −80°C and stored for batched analysis. Previous studies have shown that midregional proANP is stable for 3–6 months at −20°C and NT‐proBNP is stable for 4 months at −20°C 27, 36, 37. All samples were later transported frozen to 2 accredited laboratories, 1 laboratory for analysis of proANP 31‐67 and another laboratory for analysis of NT‐proBNP.…”

“…NT‐proANP is further cleaved into 3 segments; proANP 1‐30, 31‐67, and 68‐98 24. All segments can be analyzed,3, 4, 6, 8, 17, 18, 25 but the N‐terminal segments have been found to have longer half‐lives and may, therefore, be better suited as diagnostic tools 11, 26, 27, 28, 29. Furthermore, midregional proANP, such as proANP 31‐67, has been shown to have high biological stability, potentially because of a lower risk of fragmentation compared to proANP 1‐98 27…”