Angiotensin II measurement with high-affinity monoclonal antibodies

Abstract

Selectivity for the carboxy-terminus of angiotensin II (Ang II) and the high affinity of antibodies are prerequisites for clinical assays that evaluate Ang II in the presence of Ang I. A high-affinity monoclonal antibody (Kd = 7.1 X 10(-11) mol/l) was produced and used for the measurement of plasma Ang II. C3H mice were immunized with Ang II coupled by its carboxy-terminus to thyroglobulin. Somatic cell fusion between spleen cells and SP 2/0 myeloma cells, repeated subcloning and re-injection into mice yielded ascites containing sufficient antibody at a 2 X 10(7)-fold dilution. Radioassay standard curves show 50% tracer displacement when 32 fmol unlabelled Ang II is added and 2 fmol Ang II can be detected. Cross-reactivities, taking the reactivity with Ang II as 1.00 are: Ang I 0.003, Ang (1-7) 0.00001, Ang III 1.05, Ang(3-8) 0.88 and Ang(4-8) 0.75. Fast extraction of angiotensin from 2 ml plasma by reversible adsorption to phenylsilylsilica (Bondelut PH) provides recoveries of 96-102%. During angiotensin converting enzyme inhibition with 25 mg intravenous captopril, plasma immunoreactive Ang II decreased in supine normal volunteers from 8.6 +/- 3.6 to 4.5 +/- 3.4 fmol/ml (P less than 0.01, n = 8). It thus appears that plasma immunoreactive Ang II can now be measured after a simple extraction procedure by using monoclonal antibodies.