OBJECTIVE: To develop an efficient and stable enzyme-linked immunosorbent assay (ELISA) for detecting urinary aquaporin-2 (AQP2) water channel protein. METHODS: Rat AQP2 C-terminal peptides (CELHSPQSLPRGSKA) were synthesized and linked to KLH to prepare rabbit anti-AQP2 polyclonal antibodies, and IgG of the antibodies were labeled with horseradish peroxidase (HRP). Rat models of congestive heart failure (CHF) was established by ligation of the left coronary artery, in which both direct and sandwich ELISA for urinary AQP2 detection were tested. RESULTS: Double antibody sandwich ELISA was able to detect urinary AQP2 as low as 15.625 pmol/ml with intra-and inter-assay coefficients of variance (CVs) of 4.65% and 14.05% respectively. Urinary AQP2 concentration determined by this assay showed significant positive relation to that by Western blot analysis in CHF rats. CONCLUSION: Double antibody sandwich ELISA was successfully established to detect urine AQP2 in CHF rats, which is more efficient and simpler than Western blot analysis.
OBJECTIVE: To develop an efficient and stable enzyme-linked immunosorbent assay (ELISA) for detecting urinary aquaporin-2 (AQP2) water channel protein. METHODS:RatAQP2 C-terminal peptides (CELHSPQSLPRGSKA) were synthesized and linked to KLH to prepare rabbit anti-AQP2 polyclonal antibodies, and IgG of the antibodies were labeled with horseradish peroxidase (HRP). Rat models of congestive heart failure (CHF) was established by ligation of the left coronary artery, in which both direct and sandwich ELISA for urinary AQP2 detection were tested. RESULTS: Double antibody sandwich ELISA was able to detect urinary AQP2 as low as 15.625 pmol/ml with intra-and inter-assay coefficients of variance (CVs) of 4.65% and 14.05% respectively. Urinary AQP2 concentration determined by this assay showed significant positive relation to that by Western blot analysis in CHFrats. CONCLUSION: Double antibody sandwich ELISA was successfully established to detect urine AQP2 in CHFrats, which is more efficient and simpler than Western blot analysis.