Capillary-scale monolithic immunoaffinity columns for immunoextraction with in-line laser-induced fluorescence detection

被引:40
作者
Hodgson, RJ [1 ]
Brook, MA [1 ]
Brennan, JD [1 ]
机构
[1] McMaster Univ, Dept Chem, Hamilton, ON L8S 4M1, Canada
关键词
D O I
10.1021/ac048142p
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
A bimodal meso/macroporous monolithic silica capillary column containing an entrapped antibody was prepared by a biocompatible sol-gel process and used for nanoflow immunoaffinity chromatography and immunoextraction studies. Stationary phases were prepared by combining the protein-compatible silane precursor diglycerylsilane with an aqueous solution containing 10 000 Da poly(ethylene glycol) and the antibody. An analytical method was developed that was capable of determining both the dissociation constant and binding site content for the antifluorescein antibody within the stationary phase. The assay showed that while the antibody residing in macropores was easily removed, similar to 20% of initially loaded antibody remained active and accessible after several washes, consistent with the antibody being entrapped within the mesopores of the sol-gel matrix. The dissociation constants for fluorescein binding to the anti-fluorescein antibody were similar in solution and in the meso/ macroporous silica, indicating that the entrapped antibody retained its native conformation within such a matrix. The mixture was loaded into a 250-lim-i.d. fused-silica capillary where the polymer phase separated from the silica followed by gelation of the silica. The capillary-scale immunoaffinity columns could be operated at low back pressure using a syringe pump and were capable of performing chromatographic separations that were dependent on the presence of the antibody within the stationary phase. Such columns could also be operated using in-fine laser-induced fluorescence detection. The use of the capillary-scale monolithic columns for on-column immunoextraction and preconcentration is also demonstrated.
引用
收藏
页码:4404 / 4412
页数:9
相关论文
共 73 条
[11]   Affinity and mobility of polyclonal anti-dansyl antibodies sequestered within sol-gel-derived biogels [J].
Doody, MA ;
Baker, GA ;
Pandey, S ;
Bright, FV .
CHEMISTRY OF MATERIALS, 2000, 12 (04) :1142-1147
[12]  
Dulay MT, 2002, J SEP SCI, V25, P3, DOI 10.1002/1615-9314(20020101)25:1/2<3::AID-JSSC3>3.0.CO
[13]  
2-L
[14]   Photopolymerized sol-gel monoliths for capillary electrochromatography [J].
Dulay, MT ;
Quirino, JP ;
Bennett, BD ;
Kato, M ;
Zare, RN .
ANALYTICAL CHEMISTRY, 2001, 73 (16) :3921-3926
[15]   Species-dependency in chiral-drug recognition of serum albumin studied by chromatographic methods [J].
Fitos, I ;
Visy, J ;
Simonyi, M .
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 2002, 54 (1-3) :71-84
[16]  
Flora KK, 2001, CHEM MATER, V13, P4170, DOI [10.1021/cm010155l, 10.1021/cm0101551]
[17]   Reagentless pH-based biosensing using a fluorescently-labelled dextran co-entrapped with a hydrolytic enzyme in sol-gel derived nanocomposite films [J].
Gulcev, MD ;
Goring, GLG ;
Rakic, M ;
Brennan, JD .
ANALYTICA CHIMICA ACTA, 2002, 457 (01) :47-59
[18]   CHARACTERIZATION OF THE PROTEIN-BINDING OF CHIRAL DRUGS BY HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY - INTERACTIONS OF R-IBUPROFEN AND S-IBUPROFEN WITH HUMAN SERUM-ALBUMIN [J].
HAGE, DS ;
NOCTOR, TAG ;
WAINER, IW .
JOURNAL OF CHROMATOGRAPHY A, 1995, 693 (01) :23-32
[19]  
Hage DS, 1999, CLIN CHEM, V45, P593
[20]   THEORY OF A SEQUENTIAL ADDITION COMPETITIVE-BINDING IMMUNOASSAY BASED ON HIGH-PERFORMANCE IMMUNOAFFINITY CHROMATOGRAPHY [J].
HAGE, DS ;
THOMAS, DH ;
BECK, MS .
ANALYTICAL CHEMISTRY, 1993, 65 (11) :1622-1630