Blood was drawn into EDTA tubes and stored at 4 C until processed (generally less than one hour). Blood was centrifuged at 4 C and plasma aliquoted and frozen at −80 C until assayed.
The radioimmunoassay used for CNP was as previously described 12 (link), using a commercial anti-CNP-22 antiserum (Phoenix Pharmaceuticals, Belmont, CA). Limit-of-detection for this assay is 1.0 pM (0.2 pM after sample concentration). Within- and between-assay coefficients of variation of the assay are 4.9% and 8.9% respectively, at 2.1 pM. Cross reactivity with atrial natriuretic peptide (ANP) in this assay is <0.004%. Cross reactivity with human B-type natriuretic peptide (BNP)(at 100 pM) is approximately 4%.
The radioimmunoassay used for NTproCNP was as previously described 13 (link),14 (link) with the following alterations: 100 µl of standard or sample extract was preincubated with 50 µL primary rabbit antiserum (J39) raised against synthetic human proCNP1–15 (diluted to 1:6,000) and incubated for 22–24 hours. The antiserum J39 epitope on proCNP spans amino acid residues 3–15. Fifty microliters of radiolabeled tracer (1,500 cpm) was then added. The detection limit of this assay is 1.2 pM (0.3 pM after sample concentration). Within- and between-assay coefficients of variation are 6.8% and 8.4% respectively, at 14 pM. Cross reactivity with ANP propeptide in this assay is <0.07% and with human BNP propeptide is <0.4%.
A subset of samples was also assayed for NTproCNP using a commercially available sandwich enzyme-linked immunosorbent assay (Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria, distributed in the US by ALPCO Diagnostics, Salem, NH). Manufacturer’s instructions were followed. Limit of detection for this assay was 0.55 pM.
The radioimmunoassay used for CNP was as previously described 12 (link), using a commercial anti-CNP-22 antiserum (Phoenix Pharmaceuticals, Belmont, CA). Limit-of-detection for this assay is 1.0 pM (0.2 pM after sample concentration). Within- and between-assay coefficients of variation of the assay are 4.9% and 8.9% respectively, at 2.1 pM. Cross reactivity with atrial natriuretic peptide (ANP) in this assay is <0.004%. Cross reactivity with human B-type natriuretic peptide (BNP)(at 100 pM) is approximately 4%.
The radioimmunoassay used for NTproCNP was as previously described 13 (link),14 (link) with the following alterations: 100 µl of standard or sample extract was preincubated with 50 µL primary rabbit antiserum (J39) raised against synthetic human proCNP1–15 (diluted to 1:6,000) and incubated for 22–24 hours. The antiserum J39 epitope on proCNP spans amino acid residues 3–15. Fifty microliters of radiolabeled tracer (1,500 cpm) was then added. The detection limit of this assay is 1.2 pM (0.3 pM after sample concentration). Within- and between-assay coefficients of variation are 6.8% and 8.4% respectively, at 14 pM. Cross reactivity with ANP propeptide in this assay is <0.07% and with human BNP propeptide is <0.4%.
A subset of samples was also assayed for NTproCNP using a commercially available sandwich enzyme-linked immunosorbent assay (Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria, distributed in the US by ALPCO Diagnostics, Salem, NH). Manufacturer’s instructions were followed. Limit of detection for this assay was 0.55 pM.
