Enzyme immunoassay of bradykinin.

An enzyme immunoassay of bradykinin was developed by using beta-D-galactosidase as a labeling enzyme. Bradykinin was conjugated to beta-D-galactosidase with a new coupling agent of a hetero bis-functional type, N-(m-maleimidobenzoyloxy)-succinimide (MBS). Antisera were obtained from rabbits immunized with bradykinin linked to albumins (ovalbumin or bovine serum albumin) with toluene-2,4-diisocyanate. Double antibody method was employed to separate the antibody-bound antigen from free. The enzyme activity in the precipitate was measured with a fluorogenic substrate, 4-methyl-umbelliferyl-beta-D-galactoside. This assay is based on heterogeneous competitive binding between unlabeled and labeled antigens, so that unlabeled bradykinin reduces binding of bradykinin-enzyme conjugates to the antibody. A standard inhibition curve was linear between 3 and 300 ng bradykinin/assay tube.