Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Abstract

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

Mapping of Conformational mAb Epitopes to the C Domain of Human Angiotensin I-Converting Enzyme

“…To determine how many different antigenic regions on the C domain of human ACE could be recognized by the panel of mAbs, the ability of mAbs from this set to compete with each other for binding to ACE was tested in a modified plate precipitation assay . Recombinant human soluble tACE was preincubated with a 100-fold excess of protecting antibodies, and the ability of this complex to be precipitated by test antibodies was determined in a plate precipitation assay (Figure A in ref ). On the basis of these data, we identified three distinct regions on the surface of the C domain covered by this set of mAbs (Figure ).…”

“…Diagram showing the putative spatial relationship between epitopes. This schematic representation of mAbs binding to the C domain of human ACE is based on the results of epitope specificity studies using soluble human recombinant tACE . Competitive binding is represented by overlapping circles: nonoverlapping circles indicate noncompetitive binding.…”

Functional single-cell hybridoma screening using droplet-based microfluidics

“…Heterogeneous hybridoma cell populations [4E3 and Elec-403 (13)(14)(15)18) cells in a ratio of 1∶75; 1∶1;000 or 1∶10; 000] were encapsulated into 660 pL drops at a density of 1.25 × 10 6 cells∕mL together with 1.6 ng∕mL ACE-1 (R&D Systems). The resulting emulsion was incubated off-chip for 6 h at 37°C under a 5% CO 2 atmosphere, followed by reinjection into the integrated microfluidic chip (Fig.…”

“…However, the two catalytic sites have different substrate specificities and catalytic properties as well as different affinities for competitive inhibitors (12,13). Monoclonal antibodies against this enzyme have been used in structural and functional studies of ACE-1 (14,15) and mAbs against ACE-1 have both diagnostic and therapeutic potential: ACE-1 is a target for drugs to treat hypertension and congestive heart failure (16). Here we demonstrate the enrichment of hybridoma cells secreting the ACE-1 inhibitory mAb 4E3 (14,15) from a large excess of unrelated hybridoma cells.…”

Epitope mapping of mAbs to denatured human testicular ACE (CD143)

“…In Figure 1A, the recognition of native catalytically active human tACE is compared with that of denatured (15 min at 75°C) human tACE after adsorption on plastic. mAb 4E3 was used as a positive control for the recognition of native human tACE (41, 42). Strong denaturation conditions completely abolished the recognition of the conformational epitope of mAb 4E3 but did not influence the binding of the set of mAbs to denatured epitopes (Figure 1A).…”