DIRECT AND HOMOGENEOUS IMMUNOASSAY FOR IGG ANALYSES

被引:6
作者
HUANG, PY
LEE, CS
HEATH, C
BANIK, G
机构
[1] UNIV MARYLAND,DEPT CHEM & BIOCHEM ENGN,BALTIMORE CTY CAMPUS,CATONSVILLE,MD 21228
[2] DARTMOUTH COLL,THAYER SCH ENGN,HANOVER,NH 03755
关键词
PROTEIN-A; IGG; ELECTROSTATIC POTENTIALS; DIRECT SENSING MECHANISM; HOMOGENEOUS IMMUNOASSAY;
D O I
10.1002/bit.260400807
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A concept involving the use of antibody conjugated with reporter molecules for direct sensing of subtle changes in the local electrostatic environment of an antigen-antibody complex due to the antigen binding is presented. The proposed direct sensing mechanism is studied using the protein A-immunoglobulin G (IgG) complex as the model system. The change in the local electrostatic potential and pH around the protein A-IgG complex is observed by measuring the fluorescence intensity of fluorescein molecules conjugated with protein A before and after the introduction of IgG. Factors affecting the efficiency of our direct sensing mechanism including the conjugation location of fluorescein on protein A, the conjugation ratio of fluorescein per protein A molecule, solution conditions, and the amount of protein A-fluorescein conjugate used in the experiment are investigated. The IgG2A concentrations in the cell supernatants are analyzed by using our direct sensing method and then compared with the results obtained from enzyme-linked immunosorbent assay (ELISA). The comparisons between our homogeneous assay and heterogeneous ELISA in the analysis time, analysis procedures, cost, and sensitivity are reported and discussed.
引用
收藏
页码:913 / 918
页数:6
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