NT-proCNP ELISA Assay Kit

Additional Information

Assay Background

CNP belongs to a well characterised group of natriuretic peptides like atrial natriuretic (ANP) and brain natriuretic peptide (BNP), which play various important roles as regulatory mechanisms as well as bio-markers. They participate in the regulation of blood pressure and body fluid homeostasis, modify growth and development of cardiovascular tissues and bone. ANP and BNP are cardiac hormones, secreted by the atria and ventricles, respectively, in the normal adult heart. In contrast to ANP and BNP, very little CNP is produced by cardiac tissue, but is expressed primarily in the brain, pituitary gland, vascular endothelium, kidney and female reproductive tract.

All natriuretic peptides are produced as propeptides, which are subsequently cleaved into the biologically active, C-terminal hormone and the N-terminal fragment (NT-proANP 1-98, NT-proBNP 1-76 and NT-proCNP, which length is still under discussion). Since those N-terminal fragments are much more stable, and circulate in higher amounts than the active hormones, they are easier and more reliable to be measured in serum or plasma. So we developed a sandwich immunoassay for NT-proCNP using antibodies directed against amino acids 1-19 and 30-50. The physiological role of CNP is not only studied in cardiac disease, but also in bone developmental biology, generally in bone research, renal diseases, embryonic developmental research and vascular diseases.

Assay Principle

1. All reagents and samples have to be brought to room temperature (18-26°C) before they can be used in the NT-proCNP ELISA Assay Kit.
2. Mark position for BLANK/STD/SAMPLE/CTRL (Blank/Standard/Sample/Control) on the protocol sheet.
3. Take microtiter strips out of the aluminium bag, reserve a minimum of one well as blank. Unused strips can be stored with desiccant in the aluminium bag at +4°C (2-8°C) until the expiry date.
4. Add 50 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective wells, except blank.
5. Add 200 µl CONJ (anti NT-proCNP-HRPO) into each well, except blank, swirl gently.
6. Cover tightly and incubate for 4 hours at room temperature (18-26°C) in the dark.
7. Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer), remove remaining WASHBUF by hitting plate against paper towel after the final washing step.
8. Add 200 µl SUB (Substrate) into each well.
9. Incubate for 30 min at room temperature (18-26°C) in the dark.
10. Add 50 µl STOP (Stop solution) into each well.
11. Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Standard Curve