Premium
Affinity protection chromatography for efficient labeling of antibodies for use in affinity capillary electrophoresis
Author(s) -
Attiya Said,
DickinsonLaing Terrina,
Cesarz John,
Giese Raymond D.,
Lee William E.,
Mah David,
Harrison D. Jed
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200203)23:5<750::aid-elps750>3.0.co;2-3
Subject(s) - chemistry , chromatography , affinity chromatography , monoclonal antibody , cyanogen bromide , capillary electrophoresis , polyclonal antibodies , ovalbumin , immunoassay , detection limit , antibody , enzyme , biochemistry , antigen , peptide sequence , genetics , gene , immunology , biology
Capillary electrophoresis immunoassay (CEIA) is shown to be substantially more sensitive to the antibody (Ab) reagent quality than are immunosorbent methods such as enzyme‐linked immunosorbent assays (ELISA). Cyanine 5 (Cy5)‐labeled monoclonal anti‐ovalbumin (mAb*) was inactive for CEIA of ovalbumin (Ov), yet was functional in ELISA for Ov. ELISA showed the mAb* was at least ten times less active, accounting for the poor CEIA performance. Labeled polyclonal Ab was inactive for a dye to protein ratio greater than 1.6. An affinity protection chromatography procedure (APC) was developed for Ab labeling, which avoided degradation of the Ab binding site. Ov was covalently bound to cyanogen bromide activated cellulose gel in a column, and used to capture the Ab. The coupling efficiency for Ov to the gel was 74–97%, Ab could then be bound with 95–100% efficiency, and Ab* was recovered in 50% yield following labeling on the column. This procedure was performed successfully in three different laboratories, indicating the robustness of the optimized APC synthetic method. No inactive Ab* could be detected in the APC product. The CEIA detection limit for ovalbumin using APC labeled mAb was 173 n M , when [Ab*] was fixed at 163 n M . The association constants of mAb and mAb* were determined by CEIA.