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Enzyme Immunoassay of Angiotensin I

Departments of Physiology (T.A.) Nagasaki University School of Medicine Miyazaki 889-16, Japan
Medicine (S.S., M.M., K.H.), Nagasaki University School of Medicine Miyazaki 889-16, Japan
Department of Microbiological Chemistry, Faculty of Pharmaceutical Sciences Nagasaki University (T.K.) Nagasaki 852; Japan
the Department of Biochemistry, Medical College of Miyazaki (E.I.) Miyazaki 889-16, Japan

Address requests for reprints to: Dr. Tadaomi Aikawa, Department of Physiology, Nagasaki University School of Medicine, 12–4 Sakamotomachi, Nagasaki 852, Japan.

Abstract

A very sensitive and specific enzyme immunoassay has been developed for angiotensin I. Angiotensin I was coupled to β-D-galactosidase by a novel cross-linking reagent, N- (meta-maleimidobenzoyloxy)succinimide. No decrease in the enzyme activity was observed during the coupling procedure. In the angiotensin I-β-D-galactosidase conjugate, 0.39 mol immunoreactive angiotensin I/mol enzyme were present. A competitive assay with the enzyme-labeled angiotensin I was performed. Antibody-bound and free labeled antigen were separated from each other by the second antibody method, and the enzyme activity of the former was estimated. Using this assay, angiotensin I could be detected in the range of 1.2–50 pg. The sensitivity was 4.5-fold higher than that of the usual RIA. This assay distinguished clearly angiotensin I from angiotensin II, angiotensin III, and (Sar¹, Ile⁸)-angiotensin II. The present method was applied to measure PRA in dogs; the results correlated fairy well with those obtained by the RIA (r = 0.94).

Footnotes

1The following abbreviations were used: IgG, immunoglobulin G; MBS, N-(m-maleimidobenzoyloxy)succinimide; anti-(rabbit IgG), goat antiserum to rabbit IgG; RSA, rabbit serum albumin; BSA, bovine serum albumin; anti-(angiotensin I), rabbit antiserum to angiotensin I; CIS, CIA-IRE-SORIN.

2Previously, we named this compound meta-maleimidobenzoyl N-hydroxysuccinimide ester.

Received July 5, 1979.