Development of a novel AlphaLISA ImmunoAssay for Big angiotensin‐25

• Published in: Nephrology (Carlton, Vic.) Vic.), 2021-05, Vol.26 (5), p.479-484
• Main Authors: Nagata, Sayaka, Fukuda, Akihiro, Kikuchi, Masao, Sato, Yuji, Nagatomo, Masanao, Aburaya, Junko, Fujimoto, Shouichi, Kitamura, Kazuo
• Format: Article
• Language: English
• Subjects: AlphaLISA; Amino acids; Angiotensin; Angiotensin II; Angiotensinogen; Big angiotensin‐25; Chymase; Diabetes mellitus; diabetic kidney disease; Enzyme-linked immunosorbent assay; Glycosylation; Immunoassay; Islet cells; Kidney diseases; Pancreas; renin angiotensin system; Urine
• ISSN: 1320-5358; 1440-1797
• DOI: 10.1111/nep.13845

We previously described the discovery of Big angiotensin‐25 (Bang‐25), an angiotensin‐related peptide isolated from human urine. Bang‐25 consists of the first 25 amino acids of the N‐terminus of angiotensinogen (Aogen), with N‐linked glycosylation on the 14th amino acid and a cysteine conjugated to the 18th amino acid. Bang‐25 is rapidly converted into angiotensin II (Ang II) by chymase. Because Bang‐25 is widely distributed in human tissues, including islet cells in the pancreas and podocytes in the kidney, we hypothesized that it may participate in the Ang II production system in these tissues. To test this hypothesis, we developed a specific assay for Bang‐25 and used it to examine the urinary concentrations of Bang‐25 in patients with diabetes mellitus (DM). The assay used the Amplified Luminescent Proximity Homogeneous Assay (Alpha)‐based ELISA method (AlphaLISA) of PerkinElmer Japan and included antibodies specific to the N‐terminus of Ang II and the C‐terminus of Bang‐25. The AlphaLISA ImmunoAssay specifically recognized Bang‐25 and had no cross‐reactivity with Aogen or Ang I. Bang‐25 was detected in healthy volunteers' urine samples but not in their plasma samples. In patients with DM, the urinary Bang‐25 concentration was significantly higher than in healthy volunteers. Moreover, the results indicated that the Bang‐25 concentration in the urine may offer a different perspective on disease status from that provided by urinary albumin. This assay could provide a useful tool for determining urinary Bang‐25, which may prove an important biomarker for diabetic kidney disease. SUMMARY AT A GLANCE Nagata and colleagues measure the concentration of Big angiotensin‐25 (Bang‐25) in urine by a novel Amplified Luminescent Proximity Homogeneous Assay‐based ELISA method, which uses antibodies specific to the N‐terminus of Ang II and the C‐terminus of Bang‐25. In a validation cohort, urinary Bang‐25 levels inversely correlated with kidney function and directly with urinary albumin excretion.