Development of a peptide-based sandwich ELISA for human tissue prokallikrein with no cross-reactivity from mature kallikrein.

Human tissue prokallikrein is the enzymatically inactive zymogen of a serine proteinase involved in the liberation of vasoactive kinin peptides, and it is supposed that an impaired prokallikrein-to-kallikrein conversion is closely related to certain hypertensive and inflammatory disorders. Progress in understanding the biological role of the proenzyme has been limited by the absence of an accurate assay for the kallikrein precursor. We describe a sandwich enzyme-linked immunosorbent assay to measure human tissue prokallikrein using monospecific anti-peptide antibodies raised against propeptide derivatives.

Probing human beta1- and beta2 -adrenoceptors with domain-specific fusion protein antibodies.

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta1- or beta2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta1- or beta2-adrenoceptors in a subtype- and domain-specific manner.

Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies.

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner.

Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies.

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised.

Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies.

The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions.