Interactions of Two Monoclonal Antibodies with BNP: High Resolution Epitope Mapping Using Fluorescence Correlation Spectroscopy
详细信息 查看全文
- 作者：Sergey Y. Tetin ; Qiaoqiao Ruan ; Sylvia C. Saldana ; Mark R. Pope ; Yan Chen ; Huaiqin Wu ; Mary S. Pinkus ; Jianjun Jiang ; Paul L. Richardson
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：November 28, 2006
- 年：2006
- 卷：45
- 期：47
- 页码：14155 - 14165
- 全文大小：146K
- 年卷期：v.45,no.47(November 28, 2006)
- ISSN：1520-4995
文摘
Structure-function studies of antibody-antigen systems include the identification of aminoacid residues in the antigen that interact with an antibody and elucidation of their individual contributionsto binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesisto characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. HumanBNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteinesin positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker.We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNPalanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributionsof amino acid residues included in the continuous antigenic epitopes that are recognized by two differentmonoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of theadvantages of solution phase measurements, including high sensitivity, simplicity of manipulation withreagents, and elimination of solid phase interferences or separation steps. Significant differences in themolecular masses of the free and antibody bound BNP results in a substantial (~2.5-times) increase inthe diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusionrates of the ligand at the single molecule level introduces the ultimate opportunity for researching systemswhere the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of theligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP andbind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnosticassays on AxSYM, IMx, and Architect platforms.