Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

• Published in: Scientific reports 2023-11, Vol.13 (1), p.17565-17565, Article 17565
• Main Authors: Vear, Anika, Thalmann, Claudia, Youngs, Kristina, Hannan, Natalie, Gaspari, Tracey, Chai, Siew Yeen
• Format: Article
• Language: English
• Subjects: 631/154/53; 631/1647/664/1467; 631/1647/664/2228; 631/1647/664/2229; 631/45/607/468; Aminopeptidase; Aminopeptidases; Antibodies; Biomarkers; Blotting, Western; Cystinyl Aminopeptidase - metabolism; Enzyme-Linked Immunosorbent Assay; Enzymes; Female; Humanities and Social Sciences; Humans; Insulin; Ischemia; Metabolic disorders; multidisciplinary; Pregnancy; Science; Science (multidisciplinary); Western blotting
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-44038-1

There is growing interest in the use of the enzyme, insulin regulated aminopeptidase (IRAP), as a biomarker for conditions such as cardio-metabolic diseases and ischemic stroke, with upregulation in its tissue expression in these conditions. However, quantification of circulating IRAP has been hampered by difficulties in detecting release of the truncated, soluble form of this enzyme into the blood stream. The current study aimed to develop a sandwich ELISA using novel antibodies directed towards the soluble portion of IRAP (sIRAP), to improve accuracy in detection and quantification of low levels of sIRAP in plasma. A series of novel anti-IRAP antibodies were developed and found to be highly specific for sIRAP in Western blots. A sandwich ELISA was then optimised using two distinct antibody combinations to detect sIRAP in the low nanogram range (16–500 ng/ml) with a sensitivity of 9 ng/ml and intra-assay variability